Surface display of whole antibodies in eukaryotes

ABSTRACT

Methods for display of recombinant whole immunoglobulins or immunoglobulin libraries on the surface of eukaryote host cells, including yeast and filamentous fungi, are described. The methods are useful for screening libraries of recombinant immunoglobulins in eukaryote host cells to identify immunoglobulins that are specific for an antigen of interest.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to methods for display of wholeimmunoglobulins or libraries of immunoglobulins on the surface ofeukaryote host cells, including mammalian, plant, yeast, and filamentousfungal cells. The methods are useful for screening libraries ofeukaryotic host cells that produce recombinant immunoglobulins toidentify particular immunoglobulins with desired properties. The methodsare particularly useful for screening immunoglobulin libraries ineukaryote host cells to identify host cells that express animmunoglobulin of interest at high levels, as well as host cells thatexpress immunoglobulins that have high affinity for specific antigens.

(2) Description of Related Art

The discovery of monoclonal antibodies has evolved from hybridomatechnology for producing the antibodies to direct selection ofantibodies from human cDNA or synthetic DNA libraries. This has beendriven in part by the desire to engineer improvements in bindingaffinity and specificity of the antibodies to improve efficacy of theantibodies. Thus, combinatorial library screening and selection methodshave become a common tool for altering the recognition properties ofproteins (Ellman et al., Proc. Natl. Acad. Sci. USA 94: 2779-2782(1997): Phizicky & Fields, Microbiol. Rev. 59: 94-123 (1995)). Theability to construct and screen antibody libraries in vitro promisesimproved control over the strength and specificity of antibody-antigeninteractions.

The most widespread technique for constructing and screening antibodylibraries is phage display, whereby the protein of interest is expressedas a polypeptide fusion to a bacteriophage coat protein and subsequentlyscreened by binding to immobilized or soluble biotinylated ligand.Fusions are made most commonly to a minor coat protein, called the geneIII protein (pIII), which is present in three to five copies at the tipof the phage. A phage constructed in this way can be considered acompact genetic “unit”, possessing both the phenotype (binding activityof the displayed antibody) and genotype (the gene coding for thatantibody) in one package. Phage display has been successfully applied toantibodies, DNA binding proteins, protease inhibitors, short peptides,and enzymes (Choo & Klug, Curr. Opin. Biotechnol. 6: 431-436 (1995);Hoogenboom, Trends Biotechnol. 15: 62-70 (1997); Ladner, TrendsBiotechnol. 13: 426-430 (1995); Lowman et al., Biochemistry 30:10832-10838 (1991); Markland et al., Methods Enzymol. 267: 28-51 (1996);Matthews & Wells, Science 260: 1113-1117 (1993); Wang et al., MethodsEnzymol. 267: 52-68 (1996)).

Antibodies possessing desirable binding properties are selected bybinding to immobilized antigen in a process called “panning” Phagebearing nonspecific antibodies are removed by washing, and then thebound phage are eluted and amplified by infection of E. coli. Thisapproach has been applied to generate antibodies against many antigens.

Nevertheless, phage display possesses several shortcomings. Althoughpanning of antibody phage display libraries is a powerful technology, itpossesses several intrinsic difficulties that limit its wide-spreadsuccessful application. For example, some eukaryotic secreted proteinsand cell surface proteins require post-translational modifications suchas glycosylation or extensive disulfide isomerization, which areunavailable in bacterial cells. Furthermore, the nature of phage displayprecludes quantitative and direct discrimination of ligand bindingparameters. For example, very high affinity antibodies (Kd≦1 nM) aredifficult to isolate by panning, since the elution conditions requiredto break a very strong antibody-antigen interaction are generally harshenough (e.g., low pH, high salt) to denature the phage particlesufficiently to render it non-infective.

Additionally, the requirement for physical immobilization of an antigento a solid surface produces many artifactual difficulties. For example,high antigen surface density introduces avidity effects which mask trueaffinity. Also, physical tethering reduces the translational androtational entropy of the antigen, resulting in a smaller DS uponantibody binding and a resultant overestimate of binding affinityrelative to that for soluble antigen and large effects from variabilityin mixing and washing procedures lead to difficulties withreproducibility. Furthermore, the presence of only one to a fewantibodies per phage particle introduces substantial stochasticvariation, and discrimination between antibodies of similar affinitybecomes impossible. For example, affinity differences of six-fold orgreater are often required for efficient discrimination (Riechmann &Weill, Biochem. 32: 8848-55 (1993)). Finally, populations can beovertaken by more rapidly growing wild-type phage. In particular, sincepIII is involved directly in the phage life cycle, the presence of someantibodies or bound antigens will prevent or retard amplification of theassociated phage.

Additional bacterial cell surface display methods have been developed(Francisco, et al., Proc. Natl. Acad. Sci. USA 90: 10444-10448 (1993);Georgiou et al., Nat. Biotechnol. 15: 29-34 (1997)). However, use of aprokaryotic expression system occasionally introduces unpredictableexpression biases (Knappik & Pluckthun, Prot. Eng. 8: 81-89 (1995);Ulrich et al., Proc. Natl. Acad. Sci. USA 92: 11907-11911 (1995); Walker& Gilbert, J. Biol. Chem. 269: 28487-28493 (1994)) and bacterialcapsular polysaccharide layers present a diffusion barrier thatrestricts such systems to small molecule ligands (Roberts, Annu Rev.Microbiol. 50: 285-315 (1996)). E. coli possesses a lipopolysaccharidelayer or capsule that may interfere sterically with macromolecularbinding reactions. In fact, a presumed physiological function of thebacterial capsule is restriction of macromolecular diffusion to the cellmembrane, in order to shield the cell from the immune system (DiRienzoet al., Ann. Rev. Biochem. 47: 481-532, (1978)). Since the periplasm ofE. coli has not evolved as a compartment for the folding and assembly ofantibody fragments, expression of antibodies in E. coli has typicallybeen very clone dependent, with some clones expressing well and othersnot at all. Such variability introduces concerns about equivalentrepresentation of all possible sequences in an antibody libraryexpressed on the surface of E. coli. Moreover, phage display does notallow some important posttranslational modifications such asglycosylation that can affect specificity or affinity of the antibody.About a third of circulating monoclonal antibodies contain one or moreN-linked glycans in the variable regions. In some cases it is believedthat these N-glycans in the variable region may play a significant rolein antibody function.

The efficient production of monoclonal antibody therapeutics would befacilitated by the development of alternative test systems that utilizelower eukaryotic cells, such as yeast cells. The structural similaritiesbetween B-cells displaying antibodies and yeast cells displayingantibodies provide a closer analogy to in vivo affinity maturation thanis available with filamentous phage. In particular, because lowereukaryotic cells are able to produce glycosylated proteins, whereasfilamentous phage cannot, monoclonal antibodies produced in lowereukaryotic host cells are more likely to exhibit similar activity inhumans and other mammals as they do in test systems which utilize lowereukaryotic host cells.

Moreover, the ease of growth culture and facility of geneticmanipulation available with yeast will enable large populations to bemutagenized and screened rapidly. By contrast with conditions in themammalian body, the physicochemical conditions of binding and selectioncan be altered for a yeast culture within a broad range of pH,temperature, and ionic strength to provide additional degrees of freedomin antibody engineering experiments. The development of yeast surfacedisplay system for screening combinatorial protein libraries has beendescribed.

U.S. Pat. Nos. 6,300,065 and 6,699,658 describe the development of ayeast surface display system for screening combinatorial antibodylibraries and a screen based on antibody-antigen dissociation kinetics.The system relies on transfecting yeast with vectors that express anantibody or antibody fragment fused to a yeast cell wall protein, usingmutagenesis to produce a variegated population of mutants of theantibody or antibody fragment and then screening and selecting thosecells that produce the antibody or antibody fragment with the desiredenhanced phenotypic properties. U.S. Pat. No. 7,132,273 disclosesvarious yeast cell wall anchor proteins and a surface expression systemthat uses them to immobilize foreign enzymes or polypeptides on the cellwall.

Of interest are Tamino et al, Biotechnol. Prog. 22: 989-993 (2006),which discloses construction of a Pichia pastoris cell surface displaysystem using Flo1p anchor system; Ren et al., Molec. Biotechnol.35:103-108 (2007), which discloses the display of adenoregulin in aPichia pastoris cell surface display system using the Flo1p anchorsystem; Mergler et al., Appl. Microbiol. Biotechnol. 63:418-421 (2004),which discloses display of K. lactis yellow enzyme fused to theC-terminus half of S. cerevisiae α-agglutinin; Jacobs et al., AbstractT23, Pichia Protein expression Conference, San Diego, Calif. (Oct. 8-11,2006), which discloses display of proteins on the surface of Pichiapastoris using α-agglutinin; Ryckaert et al., Abstracts BVBMB Meeting,Vrije Universiteit Brussel, Belgium (Dec. 2, 2005), which disclosesusing a yeast display system to identify proteins that bind particularlectins; U.S. Pat. No. 7,166,423, which discloses a method foridentifying cells based on the product secreted by the cells by couplingto the cell surface a capture moiety that binds the secreted product,which can then be identified using a detection means; U.S. PublishedApplication No. 2004/0219611, which discloses a biotin-avidin system forattaching protein A or G to the surface of a cell for identifying cellsthat express particular antibodies; U.S. Pat. No. 6,919,183, whichdiscloses a method for identifying cells that express a particularprotein by expressing in the cell a surface capture moiety and theprotein wherein the capture moiety and the protein form a complex whichis displayed on the surface of the cell; U.S. Pat. No. 6,114,147, whichdiscloses a method for immobilizing proteins on the surface of a yeastor fungal using a fusion protein consisting of a binding protein fusedto a cell wall protein which is expressed in the cell.

The potential applications of engineering antibodies for the diagnosisand treatment of human disease such as cancer therapy, tumor imaging,sepsis are far-reaching. For these applications, antibodies with highaffinity (i.e., Kd≦10 nM) and high specificity are highly desirable.Anecdotal evidence, as well as the a priori considerations discussedpreviously, suggests that phage display or bacterial display systems areunlikely to consistently produce antibodies of sub-nanomolar affinity.Also, antibodies identified using phage display or bacterial displaysystems may not be susceptible to commercial scale production ineukaryotic cells. To date, no system has been developed which canaccomplish such purpose, and be used.

Therefore, development of further protein expression systems based onimproved vectors and host cell lines in which effective protein displayfacilitates development of genetically enhanced cells for recombinantproduction of immunoglobulins is a desirable objective.

BRIEF SUMMARY OF THE INVENTION

One of the most powerful applications of the display system herein isits use in the arena of immunoglobulin engineering. It has been shownthat scFv antigen-binding units can be expressed on the surface of lowereukaryote host cells with no apparent loss of binding specificity andaffinity (See for example, U.S. Pat. No. 6,300,065). It has also beenshown that full-length antibodies can be captured and bound to thesurface of hybridomas and CHO cells, for example (See U.S. Pat. Nos.6,919,183 and 7,166,423). While antibodies and fragments thereof to manydiverse antigens have been successfully isolated using phage displaytechnology, there is still a need for a robust display system forproducing immunoglobulins in eukaryotic host cells and in particular,lower eukaryote host cells. It is particularly desirable to have arobust display system for producing immunoglobulins that have human-likeglycosylation patterns. Genetically engineered eukaryote cells thatproduce glycoproteins that have various human-like glycosylationpatterns have been described in U.S. Pat. No. 7,029,872 and for examplein Choi et al., Hamilton, et al., Science 313; 1441 1443 (2006); Wildtand Gerngross, Nature Rev. 3: 119-128 (2005); Bobrowicz et al.,GlycoBiol. 757-766 (2004); Li et al., Nature Biotechnol. 24: 210-215(2006); Chiba et al., J. Biol. Chem. 273: 26298-26304 (1998); and, Maraet al., Glycoconjugate J. 16: 99-107 (1999).

The methods disclosed herein are particularly suited for thisapplication because it allows presentation of a vast diverse repertoireof full-sized immunoglobulins having particular glycosylation patternson the surface of the cell when the host cells have been geneticallyengineered to have altered or modified glycosylation pathways. In manyrespects the subject display system mimics the natural immune system.Antigen-driven stimulation can be achieved by selecting forhigh-affinity binders from a display library of cloned antibody H and Lchains. The large number of chain permutations that occur duringrecombination of H and L chain genes in developing B cells can bemimicked by shuffling the cloned H and L chains as DNA, and protein andthrough the use of site-specific recombination (Geoffory et al. Gene151: 109-113 (1994)). The somatic mutation can also be matched by theintroduction of mutations in the CDR regions of the H and L chains.

Immunoglobulins with desired binding specificity or affinity can beidentified using a form of affinity selection known as “panning”(Parmley & Smith, Gene 73:305-318 (1988)). The library ofimmunoglobulins is first incubated with an antigen of interest followedby the capture of the antigen with the bound immunoglobulins. Theimmunoglobulins recovered in this manner can then be amplified and againgain selected for binding to the antigen, thus enriching for thoseimmunoglobulins that bind the antigen of interest. One or more rounds ofselection will enable isolation of antibodies or fragments thereof withthe desired specificity or avidity. Thus, rare host cells expressing adesired antibody or fragment thereof can easily be selected from greaterthan 10⁴ different individuals in one experiment. The primary structureof the binding immunoglobulins is then deduced by nucleotide sequence ofthe individual host cell clone. When human VH and VL regions areemployed in the displayed immunoglobulins, the subject display systemsallow selection of human immunoglobulins without further manipulation ofa non-human immunoglobulins.

Therefore, in one embodiment, provided is a method for producingeukaryotic host cells that express an immunoglobulin of interest,comprising providing host cells that include a first nucleic acidmolecule encoding a capture moiety comprising a cell surface anchoringprotein fused to a binding moiety that is capable of specificallybinding an immunoglobulin operably linked to a first regulatablepromoter; transfecting the host cells with a plurality of nucleic acidmolecules encoding a genetically diverse population of heavy and lightchains of an immunoglobulin wherein at least one of the heavy or lightchain encoding nucleic acid molecules is operably linked to a secondregulatable promoter to produce a plurality of genetically diverse hostcells capable of displaying an immunoglobulin on the surface thereof;inducing expression of the first nucleic acid molecule encoding thecapture moiety for a time sufficient to produce the capture moiety onthe surface of the host cells; and inhibiting expression of the firstnucleic acid molecule encoding the capture moiety and inducingexpression of the nucleic acid molecules encoding the immunoglobulins inthe host cells to produce the host cells, which display theimmunoglobulin of interest on the surface of the cells. In furtheraspects, the method further includes contacting the host cells with adetection means that specifically binds to the immunoglobulin ofinterest displayed on the surface thereof; and isolating host cells inwhich the detection means is bound to select the host cells that expressthe immunoglobulin of interest.

In another embodiment, provided is a method for producing eukaryotichost cells that express an immunoglobulin of interest comprisingproviding a host cell that includes a first nucleic acid moleculeencoding a capture moiety comprising a cell surface anchoring proteinfused to a binding moiety that is capable of specifically binding animmunoglobulin operably linked to a first regulatable promoter;transfecting the host cell with one or more second nucleic acidmolecules encoding an immunoglobulin wherein either the moleculesencoding the light chain or the heavy chain are operably linked to asecond regulatable promoter, wherein mutagenesis is used to generate aplurality of host cells encoding a variegated population of mutants ofthe immunoglobulin; inducing expression of the capture moiety for a timesufficient to produce the capture moiety on the surface of the hostcells; inhibiting expression of the capture moiety and inducingexpression of the variegated population of mutants of the immunoglobulinin the host cells; contacting the plurality of host cells with adetection means that binds to the immunoglobulin of interest to identifyhost cells in the plurality of host cells that display theimmunoglobulin of interest on the surface thereof. In furtherembodiments, the method further includes isolating the host cells thatdisplay the immunoglobulin of interest on the surface of thereof toproduce the host cells expressing the immunoglobulin of interest.

In a further embodiment, provided is a method for producing eukaryotichost cells that express an immunoglobulin of interest, comprising:providing a host cell that includes a first nucleic acid moleculeencoding a capture moiety comprising a cell surface anchoring proteinfused to a binding moiety that is capable of specifically binding animmunoglobulin operably linked to a first regulatable promoter;transfecting the host cells with a one or more nucleic acid moleculesencoding the heavy and light chains of an immunoglobulin wherein atleast one of the heavy or light chain encoding nucleic acid molecules isoperably linked to a second regulatable promoter to generate a pluralityof host cells encoding a variegated population of mutants of theimmunoglobulins; inducing expression of the capture moiety for a timesufficient to produce the capture moiety on the surface of the hostcells; and inhibiting expression of the capture moiety and inducingexpression of the variegated population of mutants of the immunoglobulinin the host cells to produce the host cells. In further embodiments, themethod further includes contacting the host cells with a detection meansthat binds to the immunoglobulin of interest to identify host cells thatdisplay the immunoglobulin of interest on the surface thereof; andisolating the host cells that display the immunoglobulin of interest onthe surface of thereof to produce the host cells that express theimmunoglobulin of interest.

In a further embodiment, provided is a method for producing eukaryotichost cells that express an immunoglobulin of interest, comprisingproviding host cells that include a first nucleic acid molecule encodinga capture moiety comprising a cell surface anchoring protein fused to abinding moiety that is capable of specifically binding an immunoglobulinoperably linked to a first regulatable promoter; transfecting the hostcells with a plurality of nucleic acid molecules comprising open readingframes (ORFs) encoding a genetically diverse population of heavy andlight chains of an immunoglobulin wherein at least the ORFs encoding theheavy chain are operably linked to a second regulatable promoter whenthe capture moiety binds the heavy chain or at least the ORFs encodingthe light chain are operably linked to a second regulatable promoterwhen the capture moiety binds the light chain to produce a plurality ofgenetically diverse host cells capable of displaying an immunoglobulinon the surface thereof; inducing expression of the nucleic acid moleculeencoding the capture moiety for a time sufficient to produce the capturemoiety on the surface of the host cell; and inhibiting expression of thenucleic acid molecule encoding the capture moiety and inducingexpression of the nucleic acid molecules encoding the immunoglobulins inthe host cells to produce the host cells. In further embodiments, themethod further includes contacting the host cells with a detection meansthat specifically binds to the immunoglobulin of interest displayed onthe cell surface of the host cells; and isolating host cells in whichthe detection means is bound to produce the host cells that express theimmunoglobulin of interest.

In a further embodiment, provided is a method of producing eukaryotehost cells that produce an immunoglobulin having a VH domain and a VLdomain and having an antigen binding site with binding specificity foran antigen of interest, the method comprising (a) providing a library ofeukaryote host cells displaying on their surface an immunoglobulincomprising a VH domain and a VL domain, wherein the library is createdby (i) providing eukaryote host cells that express a capture moietycomprising a cell surface anchoring protein fused to a moiety capable ofbinding to an immunoglobulin wherein expression of the capture moiety iseffected by a first regulatable promoter; and (ii) transfecting the hostcells with a library of nucleic acid molecules encoding a geneticallydiverse population of immunoglobulins, wherein the VH domains of thegenetically diverse population of immunoglobulins are biased for one ormore VH gene families and wherein expression of at least one of theheavy or light chains of the immunoglobulins is effected by a secondregulatable promoter to produce a plurality of host cells, eachexpressing an immunoglobulin; (b) inducing expression of the capturemoiety in the host cells for a time sufficient to produce the capturemoiety on the surface of the host cells; (c) inhibiting expression ofthe capture moiety and inducing expression of the library of nucleicacid sequences in the host cells, whereby each host cell displays animmunoglobulin at the surface thereof to produce the host cells. Infurther embodiments, the method further includes (d) identifying hostcells in the plurality of host cells that display immunoglobulinsthereon that has a binding specificity for the antigen of interest bycontacting the plurality of host cells with the antigen of interest anddetecting the host cells that have the antigen of interest bound to theimmunoglobulin displayed thereon to produce the host cells that producethe immunoglobulin having a VH domain and a VL domain and having theantigen binding site with binding specificity for the antigen ofinterest.

In a further aspect of the above embodiment, the immunoglobulincomprises a synthetic human immunoglobulin VH domain and a synthetichuman immunoglobulin VL domain and wherein the synthetic humanimmunoglobulin VH domain and the synthetic human immunoglobulin VLdomain comprise framework regions and hypervariable loops, wherein theframework regions and first two hypervariable loops of both the VHdomain and VL domain are essentially human germ line, and wherein the VHdomain and VL domain have altered CDR3 loops. In a further aspect of theabove embodiment, in addition to having altered CDR3 loops the humansynthetic immunoglobulin VH and VL domains contain mutations in otherCDR loops. In a further still aspect of the above embodiment, each humansynthetic immunoglobulin VH domain CDR loop is of random sequence, andin a further still aspect of the above embodiment, the human syntheticimmunoglobulin VH domain CDR loops are of known canonical structures andincorporate random sequence elements.

In a further embodiment, provided is a eukaryote host cell comprising anucleic acid molecule encoding a capture moiety comprising a cellsurface anchoring protein fused to a binding moiety capable of bindingan immunoglobulin operably linked to a regulatable promoter and one ormore nucleic acid molecules encoding the heavy and light chains ofimmunoglobulins, wherein at least one of the nucleic acid moleculesencoding the heavy or light chains is operably linked to a secondregulatable promoter. In particular embodiments, the nucleic acidmolecules encoding both the heavy and light chains are operably linkedto a second regulatable promoter. In other embodiments, the nucleic acidmolecules encoding the heavy chains are operably linked to a secondregulatable promoter and the nucleic acid molecules encoding the lightchain are operably linked to a third regulatable promoter or to aconstitutive promoter. In other embodiments, the nucleic acid moleculesencoding the light chains are operably linked to a second regulatablepromoter and the nucleic acid molecules encoding the heavy chain areoperably linked to a third regulatable promoter or to a constitutivepromoter. In particular aspects, the heavy and light chains are encodedby separate open reading frames (ORFs) wherein each ORF is operablylinked to a promoter. In other aspects, the heavy and light chains areencoded by a single ORF, which produces a single fusion polypeptidecomprising the heavy and light chains in a tandem orientation, and theORF is operably linked to a regulatable promoter. The single polypeptideis cleavable between the heavy and light chains to produce separateheavy and light chain proteins, which can then associate to form afunctional antibody molecule.

In various aspects of any one of the above embodiments or aspects, thebinding moiety that binds the immunoglobulin binds the Fc region of theimmunoglobulin. Examples of such binding moieties include, but are notlimited to those selected from the group consisting of protein A,protein A ZZ domain, protein G, and protein L and fragments thereof thatretain the ability to bind to the immunoglobulin. Examples of otherbinding moieties, include but are not limited to, Fc receptor (FcR)proteins and immunoglobulin-binding fragments thereof. The FCR proteinsinclude members of the Fc gamma receptor (FcγR) family, which bind gammaimmunoglobulin (IgG), Fc epsilon receptor (FcεR) family, which bindepsilon immunoglobulin (IgE), and Fc alpha receptor (FcαR) family, whichbind alpha immunoglobulin (IgA). Particular FcR proteins that bind IgGthat can comprise the binding moiety herein include at least the IgGbinding region of FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA,FcγRIIIB, or FcγRn (neonatal).

In further aspects of any one of the above embodiments or aspects,detection means is an antigen that is capable of being bound by theimmunoglobulin of interest. In particular aspects, the antigen isconjugated to or labeled with a fluorescent moiety. In other aspects,the detection means further includes a detection immunoglobulin that isspecific for the immunoglobulin-antigen complex or is specific foranother epitope on the antigen and it is this detection immunoglobulinthat is conjugated to or labeled with a detection moiety such as afluorescent moiety.

In further aspects of any one of the above embodiments or aspects, thecell surface anchoring protein is aGlycosylphosphatidylinositol-anchored (GPI) protein. In particularaspects, the cell surface anchoring protein is selected from the groupconsisting of α-agglutinin, Cwp 1p, Cwp2p, Gas 1p, Yap3p, Flo 1p, Crh2p,Pir1p, Pir4p, Sed1p, Tip 1p, Wpip, Hpwp1p, Als3p, and Rbt5p. In furtheraspects, the cell surface anchoring protein is Sed1p.

The host cell that can be used includes both lower and high eukaryotecells. Higher eukaryote cells include mammalian, insect, and plantcells. In further aspects of any one of the above embodiments oraspects, the eukaryote is a lower eukaryote. In further aspects, thehost cell is a yeast or filamentous fungi cell, which in particularaspects is selected from the group consisting of Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichiaguercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichiasp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorphs,Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusariumvenenatum and Neurospora crassa. In particular aspects, the eukaryote isa yeast and in further aspects, the yeast is Pichia pastoris. While themethods herein have been exemplified using Pichia pastoris as the hostcell, the methods herein can be used in other lower eukaryote or highereukaryote cells for the same purposes disclosed herein.

In further aspects of any one of the aforementioned methods,O-glycosylation of glycoproteins in the host cell is controlled. Thatis, O-glycan occupancy and mannose chain length are reduced. In lowereukaryote host cells such as yeast, O-glycosylation can be controlled bydeleting the genes encoding one or more protein O-mannosyltransferases(Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase genes) (PMTs) or bygrowing the host in a medium containing one or more Pmtp inhibitors. Infurther aspects, the host cell includes a deletion of one or more of thegenes encoding PMTs and the host cell is cultivated in a medium thatincludes one or more Pmtp inhibitors. Pmtp inhibitors include but arenot limited to a benzylidene thiazolidinedione. Examples of benzylidenethiazolidinediones that can be used are 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid. In further still aspects, the host cell further includes a nucleicacid that encodes an alpha-1,2-mannosidase that has a signal peptidethat directs it for secretion.

In further aspects of any one of the aforementioned methods, host cellsfurther include lower eukaryote cells (e.g., yeast such as Pichiapastoris) that are genetically engineered to eliminate glycoproteinshaving α-mannosidase-resistant N-glycans by deleting or disrupting oneor more of the β-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, andBMT4)(See, U.S. Published Patent Application No. 2006/0211085) orabrogating translation of RNAs encoding one or more of theβ-mannosyltransferasesusing interfering RNA, antisense RNA, or the like.

In further aspects of any one of the methods herein, the host cells canfurther include lower eukaryote cells (e.g., yeast such as Pichiapastoris) that are genetically engineered to eliminate glycoproteinshaving phosphomannose residues by deleting or disrupting one or both ofthe phosphomannosyl transferase genes PNO1 and MNN4B (See for example,U.S. Pat. Nos. 7,198,921 and 7,259,007), which in further aspects canalso include deleting or disrupting the MNN4A gene or abrogatingtranslation of RNAs encoding one or more of thephosphomannosyltransferases using interfering RNA, antisense RNA, or thelike.

In further still aspects, the host cell has been genetically modified toproduce glycoproteins that have predominantly an N-glycan selected fromthe group consisting of complex N-glycans, hybrid N-glycans, and highmannose N-glycans wherein complex N-glycans are selected from the groupconsisting of Man₃GlcNAc₂, GlcNAC₍₁₋₄₎Man₃GlcNAc₂,Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂, and NANA₍₁₋₄₎Gal₍₁₋₄₎Man₃GlcNAc₂; hybridN-glycans are selected from the group consisting of Man₅GlcNAc₂,GlcNAcMan₅GlcNAc₂, GalGlcNAcMan₅GlcNAc₂, and NANAGalGlcNAcMan₅GlcNAc₂;and high Mannose N-glycans are selected from the group consisting ofMan₆GlcNAc₂, Man₇GlcNAc₂, Man₈GlcNAc₂, and Man₉GlcNAc₂.

In any one of the above embodiments or aspects, the first regulatablepromoter is a promoter that is inducible without inducing expression ofthe second regulatable promoter. The second regulatable promoter is apromoter that is inducible without inducing the expression of the firstregulatable promoter. In further aspects, the inducer of the secondregulatable promoter inhibits transcription from the first regulatablepromoter. In particular aspects in which the host cells are yeast, thefirst regulatable promoter is the GUT1 promoter and the secondregulatable promoter is the GADPH promoter. In other aspects, the firstregulatable promoter is the PCK1 promoter and the second regulatablepromoter is the GADPH promoter.

In general, in the above embodiments or aspects, the immunoglobulin willbe an IgG molecule and can include IgG1, IgG2, IgG3, and IgG4immunoglobulins and subspecies thereof. However, in particular aspectsof the above, the immunoglobulin is selected from the group consistingof IgA, IgM, IgE, camel heavy chain, and llama heavy chain.

The information derived from the host cells and methods herein can beused to produce affinity matured immunoglobulins, derivatives of theantibodies, and modified immunoglobulins or the nucleic acid encodingthe desired immunoglobulin can be subcloned into another host cell forproduction or affinity maturation of the immunoglobulin. Therefore,further provided is a host cell that expresses an immunoglobulin thathad been identified using any one of the aforementioned methods but doesnot necessarily have to be the host cell that was used to identify theimmunoglobulin. The host cell can be a prokaryote or eukaryote hostcell.

Further provided is an immunoglobulin produced by any one of the aboveembodiments or aspects.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, e.g., one thatis attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues in the Golgi apparatus for N-linked glycoproteins.

N-glycans have a common pentasaccharide core of Man3GlcNAc2 (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ withrespect to the number of branches (antennae) comprising peripheralsugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are addedto the Man3GlcNAc2 (“Man3”) core structure which is also referred to asthe “trimannose core”, the “pentasaccharide core” or the “paucimannosecore”. N-glycans are classified according to their branched constituents(e.g., high mannose, complex or hybrid). A “high mannose” type N-glycanhas five or more mannose residues. A “complex” type N-glycan typicallyhas at least one GlcNAc attached to the 1,3 mannose arm and at least oneGlcNAc attached to the 1,6 mannose arm of a “trimannose” core. ComplexN-glycans may also have galactose (“Gal”) or N-acetylgalactosamine(“GalNAc”) residues that are optionally modified with sialic acid orderivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminicacid and “Ac” refers to acetyl). Complex N-glycans may also haveintrachain substitutions comprising “bisecting” GlcNAc and core fucose(“Fuc”). Complex N-glycans may also have multiple antennae on the“trimannose core,” often referred to as “multiple antennary glycans.” A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core. The various N-glycans are alsoreferred to as “glycoforms.”

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, or “glycanase” or “glucosidase” which all refer to peptideN-glycosidase F (EC 3.2.2.18).

The term “operably linked” expression control sequences refers to alinkage in which the expression control sequence is contiguous with thegene of interest to control the gene of interest, as well as expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

The term “expression control sequence” or “regulatory sequences” areused interchangeably and as used herein refer to polynucleotidesequences which are necessary to affect the expression of codingsequences to which they are operably linked. Expression controlsequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (“expression host cell”, “expressionhost system”, “expression system” or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “transfect”, transfection”, “transfecting” and the like referto the introduction of a heterologous nucleic acid into eukaryote cells,both higher and lower eukaryote cells. Historically, the term“transformation” has been used to describe the introduction of a nucleicacid into a yeast or fungal cell; however, herein the term“transfection” is used to refer to the introduction of a nucleic acidinto any eukaryote cell, including yeast and fungal cells.

The term “eukaryotic” refers to a nucleated cell or organism, andincludes insect cells, plant cells, mammalian cells, animal cells andlower eukaryotic cells.

The term “lower eukaryotic cells” includes yeast and filamentous fungi.Yeast and filamentous fungi include, but are not limited to Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria,Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenulapolymorphs, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichodermareesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum,Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichiasp., any Saccharomyces sp., Hansenula polymorphs, any Kluyveromyces sp.,Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp. and Neurospora crassa.

As used herein, the terms “antibody,” “immunoglobulin,”“immunoglobulins” and “immunoglobulin molecule” are usedinterchangeably. Each immunoglobulin molecule has a unique structurethat allows it to bind its specific antigen, but all immunoglobulinshave the same overall structure as described herein. The basicimmunoglobulin structural unit is known to comprise a tetramer ofsubunits. Each tetramer has two identical pairs of polypeptide chains,each pair having one “light” chain (about 25 kDa) and one “heavy” chain(about 50-70 kDa). The amino-terminal portion of each chain includes avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The carboxy-terminal portion ofeach chain defines a constant region primarily responsible for effectorfunction. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively.

The light and heavy chains are subdivided into variable regions andconstant regions (See generally, Fundamental Immunology (Paul, W., ed.,2nd ed. Raven Press, N.Y., 1989), Ch. 7. The variable regions of eachlight/heavy chain pair form the antibody binding site. Thus, an intactantibody has two binding sites. Except in bifunctional or bispecificantibodies, the two binding sites are the same. The chains all exhibitthe same general structure of relatively conserved framework regions(FR) joined by three hypervariable regions, also called complementaritydetermining regions or CDRs. The CDRs from the two chains of each pairare aligned by the framework regions, enabling binding to a specificepitope. The terms include naturally occurring forms, as well asfragments and derivatives. Included within the scope of the term areclasses of immunoglobulins (Igs), namely, IgG, IgA, IgE, IgM, and IgD.Also included within the scope of the terms are the subtypes of IgGs,namely, IgG1, IgG2, IgG3, and IgG4. The term is used in the broadestsense and includes single monoclonal antibodies (including agonist andantagonist antibodies) as well as antibody compositions which will bindto multiple epitopes or antigens. The terms specifically covermonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (for example, bispecificantibodies), and antibody fragments so long as they contain or aremodified to contain at least the portion of the CH2 domain of the heavychain immunoglobulin constant region which comprises an N-linkedglycosylation site of the CH2 domain, or a variant thereof. Includedwithin the terms are molecules comprising only the Fc region, such asimmunoadhesins (U.S. Published Patent Application No. 20040136986), Fcfusions, and antibody-like molecules.

The term “Fc” fragment refers to the ‘fragment crystallized’ C-terminalregion of the antibody containing the CH2 and CH3 domains. The term“Fab” fragment refers to the ‘fragment antigen binding’ region of theantibody containing the VH, CH1, VL and CL domains.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each mAb is directed against a single determinant on theantigen. In addition to their specificity, monoclonal antibodies areadvantageous in that they can be synthesized by hybridoma culture,uncontaminated by other immunoglobulins. The term “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., (1975) Nature, 256:495, or may be made by recombinant DNAmethods (See, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.).

The term “fragments” within the scope of the terms “antibody” or“immunoglobulin” include those produced by digestion with variousproteases, those produced by chemical cleavage and/or chemicaldissociation and those produced recombinantly, so long as the fragmentremains capable of specific binding to a target molecule. Among suchfragments are Fc, Fab, Fab′, Fv, F(ab′)2, and single chain Fv (scFv)fragments. Hereinafter, the term “immunoglobulin” also includes the term“fragments” as well.

Immunoglobulins further include immunoglobulins or fragments that havebeen modified in sequence but remain capable of specific binding to atarget molecule, including: interspecies chimeric and humanizedantibodies; antibody fusions; heteromeric antibody complexes andantibody fusions, such as diabodies (bispecific antibodies),single-chain diabodies, and intrabodies (See, for example, IntracellularAntibodies: Research and Disease Applications, (Marasco, ed.,Springer-Verlag New York, Inc., 1998).

The term “catalytic antibody” refers to immunoglobulin molecules thatare capable of catalyzing a biochemical reaction. Catalytic antibodiesare well known in the art and have been described in U.S. PatentApplication Nos. 7205136; 4888281; 5037750 to Schochetman et al., U.S.Pat. Nos. 5,733,757; 5,985,626; and 6,368,839 to Barbas, I I I et al.

As used herein, the term “consisting essentially of” will be understoodto imply the inclusion of a stated integer or group of integers; whileexcluding modifications or other integers which would materially affector alter the stated integer. With respect to species of N-glycans, theterm “consisting essentially of” a stated N-glycan will be understood toinclude the N-glycan whether or not that N-glycan is fucosylated at theN-acetylglucosamine (GlcNAc) which is directly linked to the asparagineresidue of the glycoprotein.

As used herein, the term “predominantly” or variations such as “thepredominant” or “which is predominant” will be understood to mean theglycan species that has the highest mole percent (%) of total neutralN-glycans after the glycoprotein has been treated with PNGase andreleased glycans analyzed by mass spectroscopy, for example, MALDI-TOFMS or HPLC. In other words, the phrase “predominantly” is defined as anindividual entity, such as a specific glycoform, is present in greatermole percent than any other individual entity. For example, if acomposition consists of species A in 40 mole percent, species B in 35mole percent and species C in 25 mole percent, the composition comprisespredominantly species A, and species B would be the next mostpredominant species. Some host cells may produce compositions comprisingneutral N-glycans and charged N-glycans such as mannosylphosphate.Therefore, a composition of glycoproteins can include a plurality ofcharged and uncharged or neutral N-glycans. In the present invention, itis within the context of the total plurality of neutral N-glycans in thecomposition in which the predominant N-glycan determined. Thus, as usedherein, “predominant N-glycan” means that of the total plurality ofneutral N-glycans in the composition, the predominant N-glycan is of aparticular structure.

As used herein, the term “essentially free of” a particular sugarresidue, such as fucose, or galactose and the like, is used to indicatethat the glycoprotein composition is substantially devoid of N-glycanswhich contain such residues. Expressed in terms of purity, essentiallyfree means that the amount of N-glycan structures containing such sugarresidues does not exceed 10%, and preferably is below 5%, morepreferably below 1%, most preferably below 0.5%, wherein the percentagesare by weight or by mole percent. Thus, substantially all of theN-glycan structures in a glycoprotein composition according to thepresent invention are free of fucose, or galactose, or both.

As used herein, a glycoprotein composition “lacks” or “is lacking” aparticular sugar residue, such as fucose or galactose, when nodetectable amount of such sugar residue is present on the N-glycanstructures at any time. For example, in preferred embodiments of thepresent invention, the glycoprotein compositions are produced by lowereukaryotic organisms, as defined above, including yeast (for example,Pichia sp.; Saccharomyces sp.; Kluyveromyces sp.; Aspergillus sp.), andwill “lack fucose,” because the cells of these organisms do not have theenzymes needed to produce fucosylated N-glycan structures. Thus, theterm “essentially free of fucose” encompasses the term “lacking fucose.”However, a composition may be “essentially free of fucose” even if thecomposition at one time contained fucosylated N-glycan structures orcontains limited, but detectable amounts of fucosylated N-glycanstructures as described above.

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system and the variety of responses, includingantibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC), clearance of immunocomplexes(phagocytosis), antibody production by B cells and IgG serum half-lifeare defined respectively in the following: Daeron et al., 1997, AnnuRev. Immunol. 15: 203-234; Ward and Ghetie, 1995, Therapeutic Immunol.2:77-94; Cox and Greenberg, 2001, Semin. Immunol. 13: 339-345; Heyman,2003, Immunol. Lett. 88:157-161; and Ravetch, 1997, Curr. Opin. Immunol.9: 121-125.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the general operation of the method using anembodiment wherein the immunoglobulin (Ig) light and heavy chains areseparately expressed and detection of cells that express theimmunoglobulin of interest is via a labeled antigen.

FIG. 2 illustrates the construction of plasmid vector pGLY642.

FIG. 3 illustrates the construction of plasmid vector pGLY2233

FIG. 4 illustrates the construction of plasmid vector pGFI207t.

FIG. 5 illustrates the construction of plasmid vector pGLY1162.

FIG. 6 illustrates the genealogy of some of the yeast strains used todemonstrate operation of the present invention.

FIG. 7 shows a map of plasmid vector pGLY2988.

FIG. 8 shows a map of plasmid vector pGLY3200.

FIG. 9 shows maps of plasmid vectors pGLY4136 and pGLY4124.

FIG. 10 shows maps of plasmid vectors pGLY4116 and pGLY4137.

FIG. 11 shows fluorescence microscopy results of strain yGLY4134(expresses anti-Her2 antibody), strain yGLY2696 (empty strain)transfected with pGLY4136 encoding Protein A/SED1 fusion protein, andstrain yGLY4134 (expresses anti-Her2 antibody) transfected with pGLY4136encoding Protein A/SED1 fusion protein incubated with goat anti-humanIgG (H+L)-Alexa 488.

FIG. 12 shows fluorescence microscopy results of strain yGLY2696 (emptystrain) transfected with pGLY4136 encoding the Protein A/SED1 fusionprotein incubated with anti-Her2 antibody. Goat anti-human IgG(H+L)-Alexa 488 was used for detection of anti-antibody bound to theProtein A/SED1 fusion protein anchored to the cell surface.

FIG. 13 shows fluorescence microscopy results of strain yGLY2696 (emptystrain) transfected with pGLY4136 encoding Protein A/SED1 fusionprotein, strain yGLY3920 (expresses anti-CD20 antibody) transfected withpGLY4136 encoding Protein A/SED1 fusion protein, and strain yGLY4134(expresses anti-Her2 antibody) transfected with pGLY4136 encodingProtein A/SED1 fusion protein incubated with anti-Her2 antibody. Goatanti-human IgG (H+L)-Alexa 488 was used for detection of anti-antibodybound to the Protein A/SED1 fusion protein anchored to the cell surface.

FIG. 14 shows fluorescence microscopy results of strain yGLY2696 (emptystrain) transfected with pGLY4116 encoding the FcRIII/SED1 fusionprotein incubated with anti-Her2 antibody. Goat anti-human IgG(H+L)-Alexa 488 was used for detection of anti-antibody bound to theProtein A/SED1 fusion protein anchored to the cell surface.

FIG. 15 shows maps of plasmid vectors pGLY439 and pGLY4144.

FIG. 16 shows fluorescence microscopy results of strain yGLY4134 (AOXpromoter-anti-Her2 antibody) transfected with pGLY4136 (AOXpromoter-Protein A/SED1 fusion protein), strain yGLY4134 (AOXpromoter-anti-Her2 antibody) transfected with pGLY4139 (GAPDHpromoter-Protein A/SED1 fusion protein), and strain yGLY5434(GAPDHpromoter-anti-Her2 antibody) transfected with pGLY4139 (GUT)promoter-Protein A/SED1 fusion protein). Goat anti-human IgG (H+L)-Alexa488 was used for detection of anti-antibody bound to the Protein A/SED1fusion protein anchored to the cell surface.

FIG. 17 illustrates the hypothetical expression of Protein A/SED1 fusionprotein and antibody under the control of different combinations ofpromoters.

FIG. 18 shows fluorescence microscopy results of strains yGLY5757(expresses anti-CD20 antibody under control of the GAPDH promoter) andyGLY5434 (expresses anti-Her2 antibody under control of the GAPDHpromoter), each transfected with pGLY4144 encoding Protein A/SED1 fusionprotein under the control of the GUT1 promoter. Protein A/SED1 fusionprotein expression (GUT) promoter) was induced first under glycerolconditions; then antibody expression from the GAPDH promoter was inducedunder dextrose conditions, which also inhibits expression of the ProteinA/SED1 fusion protein. Goat anti-human IgG (H+L)-Alexa 488 was used fordetection of anti-antibody bound to the Protein A/SED1 fusion proteinanchored to the cell surface.

FIG. 19 shows the results of FACS sorting of the cells shown in FIG. 18.The red line represents the negative control without co-expression ofantibody. The blue line represents colonies of anti-Her2 or anti-CD20expressing strains.

FIG. 20 shows a map of plasmid vector pGLY3033.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a protein display system that is capableof displaying diverse libraries of immunoglobulins on the surface of aeukaryote host cell. The compositions and methods are particularlyuseful for the display of collections of immunoglobulins in the contextof discovery (that is, screening) or molecular evolution protocols. Asalient feature of the method is that it provides a display system inwhich a whole, intact immunoglobulin molecule of interest can bedisplayed on the surface of a host cell without having to express theimmunoglobulin molecule of interest either as fusion protein in which itis fused to a surface anchor protein or other moiety that enablescapture of the immunoglobulin by a capture moiety bound to the cellsurface. Another feature of the method is that it enables screeningdiverse libraries of immunoglobulins in host cells for a host cell inthe library that produces an immunoglobulin of interest and then enablesthe host cell to be separated from the other host cells in the librarythat do not express the immunoglobulin of interest. Importantly, theisolated host cell can then be used for production of the immunoglobulinof interest for use in therapeutic or diagnostic applications. This isan improvement over phage and yeast display methods wherein a diverselibrary of scFV or Fab fragments are screened for a host cell thatexpresses an scFV or Fab of interest, which is then used in a series ofsteps to construct a mammalian host cell that expresses a wholeimmunoglobulin with the characteristics of the scFV or Fab of interest.These subsequent steps present the risk that the desired affinity orspecificity of an scFV or Fab that has been identified during thematuration process of converting the scFV or Fab into a wholeimmunoglobulin could be abrogated or diminished.

While current phage-based methods provide substantial library diversityand have greatly improved the processes for developing immunoglobulins,a disadvantage is that the prokaryotic host cells used to construct thelibraries do not produce N-linked glycosylated glycoproteins.Posttranslational modifications such as glycosylation can affectspecificity or affinity of the immunoglobulin. It is estimated thatabout 15-20% of circulating monoclonal antibodies derived entirely inmammalian cells contain one or more N-linked glycans in the variableregions. (Jefferis, Biotechnol Progress 21: 11-16 (2005)) In some casesit is believed that these N-glycans in the variable region may play asignificant role in immunoglobulin function. For example, both positiveand negative influences on antigen binding have been seen in antibodymolecules with variable region N-glycosylation. N-glycosylationconsensus sites added within the CDR2 region of an anti-dextran antibodywere filled with carbohydrates of varying structure and showed changesin affinity, half-life and tissue targeting in a site dependent manner(Coloma et al., The Journal of Immunologyl 62: 2162-2170 (1999)).Therefore, libraries produced and screened in prokaryotic host cellswill tend to be biased against immunoglobulin species that might haveglycosylation in the variable region. Thus, immunoglobulins that mighthave particularly desirable specificity or affinity due in whole or inpart to glycosylation of one or more sites in the variable regions willnot be identified. Conversely, antibodies identified through prokaryoticscreening methods may, when expressed in a eukaryotic host, haveglycosylation structures that unfavorably impact folding or affinity.The methods and systems herein for the first time enable libraries ofimmunoglobulins to be screened wherein the libraries include populationsof immunoglobulins that are glycosylated in the variable region. Thishas the potential effect of increasing the diversity of the library overwhat would be expected if the diversity of the library was based solelyon sequence. This improvement is expected to increase the ability todevelop immunoglobulins that have greater specificity or affinity thancurrent methods permit.

The methods and systems herein also provide another advantage overcurrent methods in that eukaryote host cells that have been geneticallyengineered to produce glycoproteins that have predominantly particularN-glycan structures can be used. The N-glycan structures include any ofthe N-glycan structures currently found on human immunoglobulins orN-glycan structures that lack features not found in glycoproteins fromhigher eukaryotes. For example, in the case of yeast, the host cells canbe genetically engineered to produce immunoglobulins wherein theN-glycans are not hypermannosylated. The host cells can be geneticallyengineered to limit the amount of O-glycosylation or to modifyO-glycosylation to resemble O-glycosylation in mammalian cells.

A significant advantage of the methods and systems is that the host cellidentified in the library to produce a desired immunoglobulin can beused without further development or manipulation of the host cell or thenucleic acid molecule encoding the immunoglobulin for production of theimmunoglobulin. That is, cultivating the host cells identified herein asexpressing the desired immunoglobulin under conditions that induceexpression of the desired immunoglobulin without inducing expression ofthe capture moiety either before, after, or at the same time: the cellssecrete the desired immunoglobulin, which can then be recovered from theculture medium using methods well known in the art. An important elementis that the immunoglobulin that is produced is a whole, intactimmunoglobulin molecule. This ability to use library cells to producewhole, intact immunoglobulins is not possible with the currentphage-based or yeast-based systems. In those systems, the nucleic acidmolecules encoding the desired Fab or scFV has to be further manipulatedto construct a nucleic acid molecule that encodes a whole, intactimmunoglobulin, which is then transfected into a mammalian cell forproduction of the whole, intact immunoglobulin. Thus, the methods andsystems herein provide significant improvements to the development andproduction of immunoglobulins for therapeutic or diagnostic purposes.

What is provided then is a method for constructing and isolating aeukaryotic host cell expressing an immunoglobulin of interest from alibrary of host cells expressing a plurality of immunoglobulins. Themethod enables the construction and selection of immunoglobulins withdesirable specificity and/or affinity properties. In general, the methodcomprises providing a host cell that comprises a first nucleic acidmolecule encoding a capture moiety comprising a cell surface anchoringprotein fused to a binding moiety that is capable of specificallybinding an immunoglobulin operably linked to a first regulatablepromoter. The host cell can be further genetically engineered to produceimmunoglobulins having particular predominant N-glycan structures.

In one aspect, the host cell is propagated in a culture to provide amultiplicity of host cells, which are then transfected with a pluralityof second nucleic acid molecules, each nucleic acid molecule encodingthe heavy and/or light chains of an immunoglobulin wherein at least thenucleic acid encoding a heavy chain is operably linked to a secondregulatable promoter when the capture moiety binds the heavy chain or atleast the nucleic acid encoding a light chain is operably linked to asecond regulatable promoter when the capture moiety binds the lightchain. This produces a plurality of host cells wherein each host cell inthe plurality of host cells capable of displaying an immunoglobulin onthe surface thereof and each host cell in the plurality of host cells iscapable of displaying a particular distinct immunoglobulin species. Ingeneral, the diversity of the host cell population in the plurality ofhost cells will depend on the diversity of the library of nucleic acidmolecules that was transfected into the host cells.

In another aspect, the host cell is propagated in a culture to provide amultiplicity of host cells, which are then transfected with one or morenucleic acid second molecules encoding the heavy and/or light chains ofan immunoglobulin wherein at least the nucleic acid encoding a heavychain is operably linked to a second regulatable promoter when thecapture moiety binds the heavy chain or at least the nucleic acidencoding a light chain is operably linked to a second regulatablepromoter when the capture moiety binds the light chain to provide amultiplicity of host cells that are capable of displaying the encodedimmunoglobulin on the surface thereof. Mutagenesis of the multiplicityof host cells is used to generate a plurality of host cells that encodea variegated population of mutants of the immunoglobulin. The diversityis dependent on the mutagenesis method used. Suitable methods formutagenesis include but are not limited to cassette mutagenesis,error-prone PCR, chemical mutagenesis, or shuffling to generate arefined repertoire of altered sequences that resemble the parent nucleicacid molecule.

In further aspects, the host cell is propagated in a culture to providea multiplicity of host cells, which are then transfected with aplurality of second nucleic acid molecules, each nucleic acid moleculeencoding the heavy and/or light chains of an immunoglobulin wherein atleast the nucleic acid encoding a heavy chain is operably linked to asecond regulatable promoter when the capture moiety binds the heavychain or at least the nucleic acid encoding a light chain is operablylinked to a second regulatable promoter when the capture moiety bindsthe light chain to produce a plurality of host cells that are capable ofdisplaying an immunoglobulin on the surface thereof. Mutagenesis is thenused to generate further increase the diversity of the plurality of hostcells that are capable of displaying an immunoglobulin on the surfacethereof.

In particular embodiments, the nucleic acid molecules encoding both theheavy and light chains are operably linked to a second regulatablepromoter. In other embodiments, the nucleic acid molecules encoding atleast one of the heavy chains are operably linked to a secondregulatable promoter and the nucleic acid molecules encoding the lightchain are operably linked to a third regulatable promoter or to aconstitutive promoter. In particular aspects, a plurality of nucleicacids encoding sub-populations of heavy chains are provided whereinexpression of each sub-population is effected by a second, third, ormore regulatable promoter such that different sub-populations can beexpressed at a particular time while other sub-populations are notexpressed at that time.

In general, the heavy and light chains are encoded by separate openreading frames (ORFs) wherein each ORF is operably linked to a promoter.However, in other aspects, the heavy and light chains are encoded by asingle ORF, which produces a single fusion polypeptide comprising theheavy and light chains in a tandem orientation, and the ORF is operablylinked to a regulatable promoter. The single polypeptide is cleavablebetween the heavy and light chains to produce separate heavy and lightchain proteins, which can then associate to form a functional antibodymolecule. (See for example, U.S. Published Application No.2006/0252096).

In any one of the above aspects, the expression of the first nucleicacid molecule encoding the capture moiety is induced for a timesufficient to produce the capture moiety and allow it to be transportedto and then bound to the surface of the host cell such that the capturemoiety is capable of binding immunoglobulin molecules as they aresecreted from the host cell. Expression of the capture moiety is thenreduced or inhibited and expression of the nucleic acid moleculesencoding the heavy and/or light chains of the immunoglobulins operablylinked to the second regulatable promoter is induced. While expressionof both the heavy and light chains can be induced, in particularaspects, the expression of the heavy chain is induced and expression ofthe light chain is constitutive. In other aspects, when the capturemoiety binds the light chain, expression of the light chain can beregulated and expression of the heavy chain can be constitutive. Thus,whether it is the heavy chain or the light chain that is captureddetermines whether it is the light chain or the heavy chain whoseexpression is regulated.

Inhibition of expression of the capture moiety can be effected by nolonger providing the inducer than induces expression of the capturemoiety, or by providing an inhibitor of the first regulatable promoterthat inhibits expression of the capture moiety, or by using an inducerof expression of the immunoglobulins heavy and/or light chains operablylinked to a second or more inducible promoter that also inhibitsexpression of the capture moiety. Inhibition can be complete repressionof expression or a reduction in expression to an amount whereinexpression of the capture moiety is such that it does not interfere withthe processing and transport of the heavy and light chains through thesecretory pathway. The expressed immunoglobulin heavy and/or lightchains are processed and transported to the cell surface via the hostcell secretory pathway where they are captured by the capture moietybound to the host cell surface for display. The plurality of host cellswith the expressed immunoglobulins displayed thereon are then screenedusing a detection means that will bind to the immunoglobulin of interestbut not to other immunoglobulins to identify the host cells that displaythe immunoglobulin of interest on the surface thereof from those hostcells that do not display the immunoglobulin of interest. Host cellsthat express and display the immunoglobulin of interest are separatedfrom the host cells that do not express and display the immunoglobulinof interest to produce a population of host cells comprising exclusivelyor enriched for the host cells displaying the immunoglobulin ofinterest. These separated host cells can be propagated and used toproduce the immunoglobulin of interest in the quantities needed for theuse intended. The nucleic acid encoding the immunoglobulin can bedetermined and an expression vector encoding the heavy and light chainsof the immunoglobulin can be constructed and used to transfect anotherhost cell, which can be a prokaryotic or eukaryotic host cell.

Detection and analysis of host cells that express the immunoglobulin ofinterest can be achieved by labeling the host cells with an antigen thatis specifically recognized by the immunoglobulin of interest. Inparticular aspects, the antigen is labeled with a detection moiety. Inother aspects the antigen is unlabeled and detection is achieved byusing a detection immunoglobulin that is labeled with a detection moietyand binds an epitope of the antigen that is not bound by theimmunoglobulin of interest. This enables selection of host cells thatproduce immunoglobulins that bind the antigen at an epitope other thanthe epitope bound by the detection immunoglobulin. In another aspect,the detection immunoglobulin is specific for the immunoglobulin-antigencomplex. Regardless of the detection means, a high occurrence of thelabel indicates the immunoglobulin of interest has desirable bindingproperties and a low occurrence of the label indicates theimmunoglobulin of interest does not have desirable binding properties.

Detection moieties that are suitable for labeling are well known in theart. Examples of detection moieties, include but are not limited to,fluorescein (FITC), Alexa Fluors such as Alexa Fuor 488 (Invitrogen),green fluorescence protein (GFP), Carboxyfluorescein succinimidyl ester(CFSE), DyLight Fluors (Thermo Fisher Scientific), HyLite Fluors(AnaSpec), and phycoerythrin. Other detection moieties include but arenot limited to, magnetic beads which are coated with the antigen ofinterest or immunoglobulins that are specific for the immunoglobulin ofinterest or immunoglobulin-antigen complex. In particular aspects, themagnetic beads are coated with anti-fluorochrome immunoglobulinsspecific for the fluorescent label on the labeled antigen orimmunoglobulin. Thus, the host cells are incubated with thelabeled-antigen or immunoglobulin and then incubated with the magneticbeads specific for the fluorescent label.

Analysis of the cell population and cell sorting of those host cellsthat display the immunoglobulin of interest based upon the presence ofthe detection moiety can be accomplished by a number of techniques knownin the art. Cells that display the immunoglobulin of interest can beanalyzed or sorted by, for example, flow cytometry, magnetic beads, orfluorescence-activated cell sorting (FACS). These techniques allow theanalysis and sorting according to one or more parameters of the cells.Usually one or multiple secretion parameters can be analyzedsimultaneously in combination with other measurable parameters of thecell, including, but not limited to, cell type, cell surface antigens,DNA content, etc. The data can be analyzed and cells that display theimmunoglobulin of interest can be sorted using any formula orcombination of the measured parameters. Cell sorting and cell analysismethods are known in the art and are described in, for example, TheHandbook of Experimental Immunology, Volumes 1 to 4, (D. N. Weir,editor) and Flow Cytometry and Cell Sorting (A. Radbruch, editor,Springer Verlag, 1992). Cells can also be analyzed using microscopytechniques including, for example, laser scanning microscopy,fluorescence microscopy; techniques such as these may also be used incombination with image analysis systems. Other methods for cell sortinginclude, for example, panning and separation using affinity techniques,including those techniques using solid supports such as plates, beads,and columns.

In further aspects, provided is a library method for identifying andselecting cells that produce an immunoglobulin having a desiredspecificity and/or affinity for a particular antigen. The methodcomprises providing a library of eukaryote host cells displaying ontheir surface an immunoglobulin comprising a VH domain and a VL domain,wherein the library is created by (i) providing eukaryote host cellsthat express a capture moiety comprising a cell surface anchoringprotein fused to a moiety capable of binding an immunoglobulin whereinexpression of the capture moiety is effected by a first regulatablepromoter; and (ii) transfecting the host cells with a library of nucleicacid sequences encoding a genetically diverse population ofimmunoglobulins, wherein the VH domains of the genetically diversepopulation of immunoglobulins are biased for one or more VH genefamilies and wherein expression of at least one or more heavy or lightchains is effected by a second regulatable promoter to produce aplurality of host cells, each host cell in the plurality of host cellsexpresses an immunoglobulin species. Expression of the capture moiety isinduced in the plurality of host cells for a time sufficient to producethe capture moiety on the surface of the host cells. Then expression ofthe of the capture moiety while expression of the library of nucleicacid sequences is induced in the plurality of host cells to produce aplurality of host cells wherein each host cell displays animmunoglobulin species at the surface thereof. Host cells in theplurality of host cells that display immunoglobulins thereon that has abinding specificity for the antigen of interest are identified bycontacting the plurality of host cells with the antigen of interest anddetecting the host cells that have the antigen of interest bound to theimmunoglobulin displayed thereon to produce the host cells that producethe immunoglobulin having a VH domain and a VL domain and having theantigen binding site with binding specificity for the antigen ofinterest. In particular aspects, the immunoglobulin comprises asynthetic human immunoglobulin VH domain and a synthetic humanimmunoglobulin VL domain and further, the synthetic human immunoglobulinVH domain and the synthetic human immunoglobulin VL domain compriseframework regions and hypervariable loops, wherein the framework regionsand first two hypervariable loops of both the VH domain and VL domainare essentially human germ line, and wherein the VH domain and VL domainhave altered CDR3 loops.

This provides a library of host cells that are capable of expressing aplurality of immunoglobulin molecules, which can be captured anddisplayed on the cell surface for detection by a detection means thatcan bind an immunoglobulin specific for a particular antigen and therebyenable the host cell expressing the immunoglobulin to be identified fromthe plurality of host cells in the library. In general, the detectionmeans will usually use the antigen that has been labeled with adetection moiety. These host cells can be isolated from the plurality ofhost cells by any means currently used for selection of particular cellsin a population of cells, e.g., FACS sorting.

Thus, the method comprises at least two components. The first componentis a helper vector that contains an expression cassette comprising thefirst nucleic acid molecule that encodes and expresses a capture moietythat in particular embodiments comprises a cell surface anchoringprotein or cell wall binding protein that is capable of binding orintegrating to the surface of the host cell fused at its N- orC-terminus to a binding moiety capable of binding an immunoglobulin. Thebinding moiety is located at the end of the cell surface anchoringprotein that is exposed to the extracellular environment such that thebinding moiety is capable of interacting with an immunoglobulin. Theimmunoglobulin binding moiety includes the immunoglobulin bindingdomains from such molecules as protein A, protein G, protein L, or thelike or an Fc receptor.

The second component is one or more vectors that contain expressioncassettes that encode and express the heavy and light chains of animmunoglobulin of interest or libraries of which the immunoglobulin ofinterest is to be selected (for example, a library of vectors expressingimmunoglobulins). In particular aspects, the nucleic acid moleculeencoding the immunoglobulin may include the nucleotide sequencesencoding both the heavy and the light chains of the immunoglobulins,e.g., an immunoglobulin having a VH domain and a VL domain and having anantigen binding site with binding specificity for an antigen ofinterest. In other aspects, the heavy and light chains are encoded onseparate nucleic acid molecules. In either case, these nucleic acidmolecules may further include when desirable codon optimizations toenhance translation of the mRNA encoding the immunoglobulins in the hostcell chosen. The nucleic acid molecule may further include whendesirable replacement of endogenous signal peptides with signal peptidesthat are appropriate for the host cell chosen.

In one aspect, the above nucleic acid molecule can comprise a singleexpression cassette operably linked to a second regulatable promoterwherein the open reading frames (ORFs) for the light and heavy chainsare in frame and separated by a nucleic acid molecule encoding in framea protease cleavage site that upon expression produces a fusion proteinthat is processed post-translationally with a protease specific for theprotease cleavage site to produce the light and heavy chains of theimmunoglobulin. Examples of these expression cassettes can be found infor example, U.S. Publication No. 20060252096. In another aspect, theheavy and light immunoglobulin chains are expressed from separateexpression cassettes wherein the ORF encoding each of the light andheavy chains is operably linked to a second regulatable promoter.Examples of these expression cassettes can be found in for example, U.S.Pat. Nos. 4,816,567 and 4,816,397. In a further aspect, the heavy andlight immunoglobulin chains are expressed from separate expressioncassettes wherein the ORF encoding the heavy chain is operably linked toa second regulatable promoter and the ORF encoding the light chain isoperably linked to a constitutive promoter.

In particular aspects, the encoded immunoglobulin comprises a synthetichuman immunoglobulin VH domain and a synthetic human immunoglobulin VLdomain and wherein the synthetic human immunoglobulin VH domain and thesynthetic human immunoglobulin VL domain comprise framework regions andhypervariable loops, wherein the framework regions and first twohypervariable loops of both the VH domain and VL domain are essentiallyhuman germ line, and wherein the VH domain and VL domain have alteredCDR3 loops. In further still aspects, in addition to having altered CDR3loops, the human synthetic immunoglobulin VH and VL domains containmutations in other CDR loops. In further aspects, each human syntheticimmunoglobulin VH domain CDR loop is of random sequence. In furtherstill aspects, the human synthetic immunoglobulin VH domain CDR loopsare of known canonical structures and incorporate random sequenceelements.

Both of the components can be provided in vectors which integrate thenucleic acid molecules into the genome of the host cell by homologousrecombination. Homologous recombination can be double crossover orsingle crossover homologous recombination. Roll-in single crossoverhomologous recombination has been described in Nett et al., Yeast 22:295-304 (2005). Each component can be integrated in the same locus inthe genome or in separate loci in the genome. Alternatively, one or bothcomponents can be transiently expressed in the host cell.

FIG. 1 illustrates the general operation of the method using anembodiment wherein the immunoglobulin light and heavy chains areseparately expressed and detection is via a labeled antigen. FIG. 1shows an expression cassette encoding the capture moiety fusion proteinoperably linked to promoter A and expression cassettes encoding theimmunoglobulin (Ig) light and heavy chains, each operably linked topromoter B. As shown, the host cell is transfected with the expressioncassettes and the transformed cells grown under conditions that induceexpression of the capture moiety fusion protein via promoter A. Thecapture moiety fusion protein is anchored to the cell surface. Then thecells are grown under conditions that inhibit or reduce expression ofthe capture moiety fusion protein but induce expression of theimmunoglobulin light and heavy chains via promoter B. Theimmunoglobulins are secreted from the cells and captured by the capturemoiety fusion protein anchored to the cell surface. The cells with thecaptured immunoglobulins are then screened for the Ig of interest usinga antigen labeled with a detection moiety. As shown, not all cells willproduce the immunoglobulin of interest. Cells that bind the labeledantigen are selected and separated from cells that do not produce theimmunoglobulin of interest. This produces cells that express theimmunoglobulin of interest. These cells can be used for producing theimmunoglobulin for use in therapeutic or diagnostic applications.Alternatively, the cells can undergo mutagenesis that introducesmutations into the expression cassettes encoding the immunoglobulins andthe cells screened for cells that produce immunoglobulins withproperties that have been modified or altered from those properties inthe immunoglobulin prior to mutagenesis and which are desired. Cellsthat express immunoglobulins having modified or altered but desiredproperties can be separated from the other cells and used for producingthe immunoglobulin for therapeutic or diagnostic applications.

Glycosylphosphatidylinositol-anchored (GPI) proteins provide a suitablemeans for tethering the capture moiety to the surface of the host cell.GPI proteins have been identified and characterized in a wide range ofspecies from humans to yeast and fungi. Thus, in particular aspects ofthe methods disclosed herein, the cell surface anchoring protein is aGPI protein or fragment thereof that can anchor to the cell surface.Lower eukaryotic cells have systems of GPI proteins that are involved inanchoring or tethering expressed proteins to the cell wall so that theyare effectively displayed on the cell wall of the cell from which theywere expressed. For example, 66 putative GPI proteins have beenidentified in Saccharomyces cerevisiae (See, de Groot et al., Yeast 20:781-796 (2003)). GPI proteins which may be used in the methods hereininclude, but are not limited to, Saccharomyces cerevisiae CWP1, CWP2,SER1, and GAS1; Pichia pastoris SP1 and GAS1; and H. polymorpha TIP1.Additional GPI proteins may also be useful. Additional suitable GPIproteins can be identified using the methods and materials of theinvention described and exemplified herein.

The selection of the appropriate GPI protein will depend on theparticular recombinant protein to be produced in the host cell and theparticular post-translation modifications to be performed on therecombinant protein. For example, production of immunoglobulins withparticular glycosylation patterns will entail the use of recombinanthost cells that produce glycoproteins having particular glycosylationpatterns. The GPI protein most suitable in a system for producingantibodies or fragments thereof that have predominantly Man₅GlcNAc₂N-glycosylation many not necessarily be the GPI protein most suitable ina system for producing antibodies or thereof having predominantlyGal₂GlcNAc₂Man₃GlcNAc₂ N-glycosylation. In addition, the GPI mostsuitable in a system for producing immunoglobulins specific for oneepitope or antigen may not necessarily be the most suitable GPI proteinin a system for producing immunoglobulins specific for another epitopeor antigen.

Therefore, further provided is a library method for constructing thehost cell that is to be used for producing a particular immunoglobulin.In general, the host cell that is desired to produce the particularimmunoglobulin is selected based on the desired characteristics thatwill be imparted to the particular immunoglobulin produced by the hostcell. For example, a host cell that produces glycoproteins havingpredominantly Man₅GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycosylation isselected and a library of vectors encoding GPI proteins fused to one ormore immunoglobulin capture moieties is then provided (GPI-IgG capturemoiety). A library of host cells is then constructed wherein each hostcell to make up the library is transfected with one of the vectors inthe library of vectors encoding GPI-IgG capture moiety fusion proteinssuch that each host cell species in the library will express oneparticular GPI-IgG capture moiety fusion protein. Each host cell speciesof the library is then transfected with a vector encoding the desiredparticular immunoglobulin. The host cell that results in the bestpresentation of the particular immunoglobulin on the surface of the hostcell is selected as the host cell for producing the particularimmunoglobulin.

In general, the GPI protein used in the methods disclosed herein is achimeric protein or fusion protein comprising the GPI protein fused atits N-terminus to the C-terminus of an immunoglobulin capture moiety.The N-terminus of the capture moiety is fused to the C-terminus of asignal sequence or peptide that enables the GPI-IgG capture moietyfusion protein to be transported through the secretory pathway to thecell surface where the GPI-IgG capture moiety fusion protein is secretedand then bound to the cell surface. In some aspects, the GPI-IgG capturemoiety fusion protein comprises the entire GPI protein and in otheraspects, the GPI-IgG capture moiety fusion protein comprises the portionof the GPI protein that is capable of binding to the cell surface.

The immunoglobulin capture moiety can comprise any molecule that canbind to an immunoglobulins. A multitude of Gram-positive bacteriaspecies have been isolated that express surface proteins with affinitiesfor mammalian immunoglobulins through interaction with their heavychains. The best known of these immunoglobulin binding proteins are type1 Staphylococcus Protein A and type 2 Streptococcus Protein G which havebeen shown to interact principally through the C2-C3 interface on the Fcregion of human immunoglobulins. In addition, both have also been shownto interact weakly to the Fab region, but again through theimmunoglobulin heavy chain.

Recently, a novel protein from Peptococcusmagnums, Protein L, has beenreported that was found to bind to human, rabbit, porcine, mouse, andrat immunoglobulins uniquely through interaction with their lightchains. In humans this interaction has been shown to occur exclusivelyto the kappa chains. Since both kappa and lambda light chains are sharedbetween different classes, Protein L binds strongly to all humanclasses, in particular to the multi-subunit IgM, and similarly isexpected to bind to all classes in species that show Protein L lightchain binding.

Examples of other binding moieties, include but are not limited to, Fcreceptor (FcR) proteins and immunoglobulin-binding fragments thereof.The FCR proteins include members of the Fc gamma receptor (FcγR) family,which bind gamma immunoglobulin (IgG), Fc epsilon receptor (FcεR)family, which bind epsilon immunoglobulin (IgE), and Fc alpha receptor(FcαR) family, which bind alpha immunoglobulin (IgA). Particular FcRproteins that bind IgG and can be used to comprise the capture moietydisclosed herein include at least the immunoglobulin binding portion ofany one of FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB, orFcγRn (neonatal).

Regulatory sequences which may be used in the practice of the methodsdisclosed herein include signal sequences, promoters, and transcriptionterminator sequences. It is generally preferred that the regulatorysequences used be from a species or genus that is the same as or closelyrelated to that of the host cell or is operational in the host cell typechosen. Examples of signal sequences include those of Saccharomycescerevisiae invertase; the Aspergillus niger amylase and glucoamylase;human serum albumin; Kluyveromyces maxianus inulinase; and Pichiapastoris mating factor and Kar2. Signal sequences shown herein to beuseful in yeast and filamentous fungi include, but are not limited to,the alpha mating factor presequence and preprosequence fromSaccharomyces cerevisiae; and signal sequences from numerous otherspecies.

Examples of promoters include promoters from numerous species, includingbut not limited to alcohol-regulated promoter, tetracycline-regulatedpromoters, steroid-regulated promoters (e.g., glucocorticoid, estrogen,ecdysone, retinoid, thyroid), metal-regulated promoters,pathogen-regulated promoters, temperature-regulated promoters, andlight-regulated promoters. Specific examples of regulatable promotersystems well known in the art include but are not limited tometal-inducible promoter systems (e.g., the yeast copper-metallothioneinpromoter), plant herbicide safner-activated promoter systems, plantheat-inducible promoter systems, plant and mammalian steroid-induciblepromoter systems, Cym repressor-promoter system (Krackeler Scientific,Inc. Albany, N.Y.), RheoSwitch System (New England Biolabs, BeverlyMass.), benzoate-inducible promoter systems (See WO2004/043885), andretroviral-inducible promoter systems. Other specific regulatablepromoter systems well-known in the art include thetetracycline-regulatable systems (See for example, Berens & Hillen, EurJ Biochem 270: 3109-3121 (2003)), RU 486-inducible systems,ecdysone-inducible systems, and kanamycin-regulatable system. Lowereukaryote-specific promoters include but are not limited to theSaccharomyces cerevisiae TEF-1 promoter, Pichia pastoris GAPDH promoter,Pichia pastoris GUT1 promoter, PMA-1 promoter, Pichia pastoris PCK-1promoter, and Pichia pastoris AOX-1 and AOX-2 promoters. For temporalexpression of the GPI-IgG capture moiety and the immunoglobulins, thePichia pastoris GUT1 promoter operably linked to the nucleic acidmolecule encoding the GPI-IgG capture moiety and the Pichia pastorisGAPDH promoter operably linked to the nucleic acid molecule encoding theimmunoglobulin are shown in the examples herein to be useful.

Examples of transcription terminator sequences include transcriptionterminators from numerous species and proteins, including but notlimited to the Saccharomyces cerevisiae cytochrome C terminator; andPichia pastoris ALG3 and PMA1 terminators.

Nucleic acid molecules encoding immunoglobulins can be obtained from anysuitable source including spleen and liver cells and antigen-stimulatedantibody producing cells, obtained from either in vivo or in vitrosources. Regardless of source, the cellular VH and VL mRNAs are reversetranscribed into VH and VL cDNA sequences. Reverse transcription may beperformed in a single step or in an optional combined reversetranscription/PCR procedure to produce cDNA libraries containing aplurality of immunoglobulin-encoding DNA molecules. (See, for example,Marks et al., J. Mol. Biol. 222: 581-596 (1991)). Nucleic acid moleculescan also be synthesized de novo based on sequences in the scientificliterature. Nucleic acid molecules can also be synthesized by extensionof overlapping oligonucleotides spanning a desired sequence (See, e.g.,Caldas et al., Protein Engineering, 13: 353-360 (2000)). Humanizedimmunoglobulin-encoding cDNA libraries can be constructed by PCRamplifying the complementary-determining regions (CDR) from the cDNAs inone or more libraries from any source and integrating the PCR amplifiedCDR-encoding nucleic acid molecules into nucleic acid molecules encodinga human immunoglobulin framework to produce a cDNA library encoding aplurality of humanized immunoglobulins (See, for example, U.S. Pat. Nos.6,180,370; 6,632,927; and, 6,872,392). Chimeric immunoglobulin-encodingcDNA libraries can be constructed by PCR amplifying the variable regionsfrom the cDNAs in the cDNA library from one species and integrating thenucleic acid molecules encoding the PCR-amplified variable regions ontonucleic acid molecules encoding immunoglobulin constant regions fromanother species to produce a cDNA library encoding a plurality ofchimeric immunoglobulins (See, for example, U.S. Pat. No. 5,843,708).Various methods that have been developed for the creation of diversitywithin protein libraries, including random mutagenesis (Daugherty etal., Proc. Natl. Acad. Sci. USA, 97: 2029-2034 (2000); Boder et al.,Proc. Natl. Acad. Sci. USA, 97: 10701-10705 (2000); Holler et al., Proc.Natl. Acad. Sci. USA, 97: 5387-5392 (2000)), in vitro DNA shuffling(Stemmer, Nature, 370: 389-391 (1994); Stemmer, Proc. Natl. Acad. Sci.USA, 91: 10747-10751 (1994)), in vivo DNA shuffling (Swers et al., Nucl.Acid Res. 32: e36 (2004)), and site-specific recombination (Rehberg etal., J. Biol. Chem., 257: 11497-11502 (1982); Streuli et al., Proc.Natl. Acad. Sci. USA, 78: 2848-2852 (1981); Waterhouse et al.,. (1993)Nucl. Acids Res., 21: 2265-2266 (1993); Sblattero & Bradbury, Nat.Biotechnol., 18: 75-80 (2000)) can be used or adapted to produce theplurality of host cells disclosed herein that express immunoglobulinsand the capture moiety comprising a cell surface anchoring protein fusedto a binding moiety that is capable of specifically binding animmunoglobulin.

Production of active immunoglobulins requires proper folding of theprotein when it is produced and secreted by the cells. In E. coli, thecomplexity and large size of an antibody presents an obstacle to properfolding and assembly of the expressed light and heavy chainpolypeptides, resulting in poor yield of intact antibody. The presenceof effective molecular chaperone proteins may be required, or mayenhance the ability of the cell to produce and secrete properly foldedproteins. The use of molecular chaperone proteins to improve productionof immunoglobulins in yeast has been disclosed in U.S. Pat. No.5,772,245; U.S. Pat. Nos. 5,700,678 and 5,874,247; U.S. ApplicationPublication No. 2002/0068325; Toman et al., J. Biol. Chem. 275:23303-23309 (2000); Keizer-Gunnink et al., Martix Biol. 19: 29-36(2000); Vad et al., J. Biotechnol. 116: 251-260 (2005); Inana et al.,Biotechnol. Bioengineer. 93: 771-778 (2005); Zhang et al., Biotechnol.Prog. 22: 1090-1095 (2006); Damasceno et al., Appl. Microbiol.Biotechnol. 74: 381-389 (2006); Huo et al., Protein Express. Purif. 54:234-239 (2007); and copending application Ser. No. 61/066,409, filed 20Feb. 2008.

As used herein, the methods can use host cells from any kind of cellularsystem which can be modified to express a capture moiety comprising acell surface anchoring protein fused to a binding moiety capable ofbinding an immunoglobulin and whole, intact immunoglobulins. Within thescope of the invention, the term “cells” means the cultivation ofindividual cells, tissues, organs, insect cells, avian cells, reptiliancells, mammalian cells, hybridoma cells, primary cells, continuous celllines, stem cells, plant cells, yeast cells, filamentous fungal cells,and/or genetically engineered cells, such as recombinant cellsexpressing and displaying a glycosylated immunoglobulin.

In a further embodiment, lower eukaryotes such as yeast or filamentousfungi are used for expression and display of the immunoglobulins becausethey can be economically cultured, give high yields, and whenappropriately modified are capable of suitable glycosylation. Yeastparticularly offers established genetics allowing for rapidtransfections, tested protein localization strategies and facile geneknock-out techniques. Suitable vectors have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase orother glycolytic enzymes, and an origin of replication, terminationsequences and the like as desired.

Host cells useful in the present invention include Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichiaguercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichiasp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha,Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusariumvenenatum and Neurospora crassa. Various yeasts, such as K. lactis,Pichia pastoris, Pichia methanolica, and Hansenula polymorphs areparticularly suitable for cell culture because they are able to grow tohigh cell densities and secrete large quantities of recombinant protein.Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp,Neurospora crassa and others can be used to produce glycoproteins of theinvention at an industrial scale. In the case of lower eukaryotes, cellsare routinely grown from between about 1.5 to 3 days under conditionsthat induce expression of the capture moiety. The induction ofimmunoglobulin expression while inhibiting expression of the capturemoiety is for about 1 to 2 days. Afterwards, the cells are analyzed forthose cells that display the immunoglobulin of interest.

Lower eukaryotes, particularly yeast and filamentous fungi, can begenetically modified so that they express glycoproteins in which theglycosylation pattern is human-like or humanized. In this manner,glycoprotein compositions can be produced in which a specific desiredglycoform is predominant in the composition. Such can be achieved byeliminating selected endogenous glycosylation enzymes and/or geneticallyengineering the host cells and/or supplying exogenous enzymes to mimicall or part of the mammalian glycosylation pathway as described in US2004/0018590. If desired, additional genetic engineering of theglycosylation can be performed, such that the glycoprotein can beproduced with or without core fucosylation. Use of lower eukaryotic hostcells is further advantageous in that these cells are able to producehighly homogenous compositions of glycoprotein, such that thepredominant glycoform of the glycoprotein may be present as greater thanthirty mole percent of the glycoprotein in the composition. Inparticular aspects, the predominant glycoform may be present in greaterthan forty mole percent, fifty mole percent, sixty mole percent, seventymole percent and, most preferably, greater than eighty mole percent ofthe glycoprotein present in the composition.

Lower eukaryotes, particularly yeast, can be genetically modified sothat they express glycoproteins in which the glycosylation pattern ishuman-like or humanized. Such can be achieved by eliminating selectedendogenous glycosylation enzymes and/or supplying exogenous enzymes asdescribed by Gerngross et al., US 20040018590. For example, a host cellcan be selected or engineered to be depleted in 1,6-mannosyl transferaseactivities, which would otherwise add mannose residues onto the N-glycanon a glycoprotein.

In one embodiment, the host cell further includes an α1,2-mannosidasecatalytic domain fused to a cellular targeting signal peptide notnormally associated with the catalytic domain and selected to target theα1,2-mannosidase activity to the ER or Golgi apparatus of the host cell.Passage of a recombinant glycoprotein through the ER or Golgi apparatusof the host cell produces a recombinant glycoprotein comprising aMan₅GlcNAc₂ glycoform, for example, a recombinant glycoproteincomposition comprising predominantly a Man₅GlcNAc₂ glycoform. Forexample, U.S. Pat. No. 7,029,872 and U.S. Published Patent ApplicationNos. 2004/0018590 and 2005/0170452 disclose lower eukaryote host cellscapable of producing a glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a GlcNAc transferase I (GnT I) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase I activity tothe ER or Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GlcNAcMan₅GlcNAc₂ glycoform, forexample a recombinant glycoprotein composition comprising predominantlya GlcNAcMan₅GlcNAc₂ glycoform. U.S. Pat. No. 7,029,872 and U.S.Published Patent Application Nos. 2004/0018590 and 2005/0170452 discloselower eukaryote host cells capable of producing a glycoproteincomprising a GlcNAcMan₅GlcNAc₂ glycoform. The glycoprotein produced inthe above cells can be treated in vitro with a hexaminidase to produce arecombinant glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a mannosidase II catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target mannosidase II activity to the ER or Golgi apparatusof the host cell. Passage of the recombinant glycoprotein through the ERor Golgi apparatus of the host cell produces a recombinant glycoproteincomprising a GlcNAcMan₃GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAcMan₃GlcNAc₂glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent ApplicationNo. 2004/0230042 discloses lower eukaryote host cells that expressmannosidase II enzymes and are capable of producing glycoproteins havingpredominantly a GlcNAc₂Man₃GlcNAc₂ glycoform. The glycoprotein producedin the above cells can be treated in vitro with a hexaminidase toproduce a recombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes GlcNAc transferase II (GnT II) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase II activityto the ER or Golgi apparatus of the host cell. Passage of therecombinant glycoprotein through the ER or Golgi apparatus of the hostcell produces a recombinant glycoprotein comprising a GlcNAc₂Man₃GlcNAc₂glycoform, for example a recombinant glycoprotein composition comprisingpredominantly a GlcNAc₂Man₃GlcNAc₂ glycoform. U.S. Pat. No. 7,029,872and U.S. Published Patent Application Nos. 2004/0018590 and 2005/0170452disclose lower eukaryote host cells capable of producing a glycoproteincomprising a GlcNAc₂Man₃GlcNAc₂ glycoform. The glycoprotein produced inthe above cells can be treated in vitro with a hexaminidase to produce arecombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a galactosyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target galactosyltransferase activity to the ERor Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GalGlcNAc₂Man₃GlcNAc₂ orGal₂GlcNAc₂Man₃GlcNAc₂ glycoform, or mixture thereof for example arecombinant glycoprotein composition comprising predominantly aGalGlcNAc₂Man₃GlcNAc₂ glycoform or Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof. U.S. Pat. No. 7,029,872 and U.S. Published PatentApplication No. 2006/0040353 discloses lower eukaryote host cellscapable of producing a glycoprotein comprising a Gal₂GlcNAc₂Man₃GlcNAc₂glycoform. The glycoprotein produced in the above cells can be treatedin vitro with a galactosidase to produce a recombinant glycoproteincomprising a GlcNAc₂Man₃GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAc₂Man₃GlcNAc₂glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a sialyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target sialytransferase activity to the ER orGolgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising predominantly aNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform or NANAGal₂GlcNAc₂Man₃GlcNAc₂glycoform or mixture thereof. For lower eukaryote host cells such asyeast and filamentous fungi, it is useful that the host cell furtherinclude a means for providing CMP-sialic acid for transfer to theN-glycan. U.S. Published Patent Application No. 2005/0260729 discloses amethod for genetically engineering lower eukaryotes to have a CMP-sialicacid synthesis pathway and U.S. Published Patent Application No.2006/0286637 discloses a method for genetically engineering lowereukaryotes to produce sialylated glycoproteins. The glycoproteinproduced in the above cells can be treated in vitro with a neuraminidaseto produce a recombinant glycoprotein comprising predominantly aGal₂GlcNAc₂Man₃GlcNAc₂ glycoform or GalGlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof.

Any one of the preceding host cells can further include one or moreGlcNAc transferase selected from the group consisting of GnT III, GnTIV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected(GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N-glycanstructures such as disclosed in U.S. Published Patent Application Nos.2004/074458 and 2007/0037248.

In further embodiments, the host cell that produces glycoproteins thathave predominantly GlcNAcMan₅GlcNAc₂ N-glycans further includes agalactosyltransferase catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target Galactosyltransferase activity to the ER or Golgiapparatus of the host cell. Passage of the recombinant glycoproteinthrough the ER or Golgi apparatus of the host cell produces arecombinant glycoprotein comprising predominantly theGalGlcNAcMan₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell thatproduced glycoproteins that have predominantly the GalGlcNAcMan₅GlcNAc₂N-glycans further includes a sialyltransferase catalytic domain fused toa cellular targeting signal peptide not normally associated with thecatalytic domain and selected to target sialytransferase activity to theER or Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a NANAGalGlcNAcMan₅GlcNAc₂glycoform.

Various of the preceding host cells further include one or more sugartransporters such as UDP-GlcNAc transporters (for example, Kluyveromyceslactis and Mus musculus UDP-GlcNAc transporters), UDP-galactosetransporters (for example, Drosophila melanogaster UDP-galactosetransporter), and CMP-sialic acid transporter (for example, human sialicacid transporter). Because lower eukaryote host cells such as yeast andfilamentous fungi lack the above transporters, it is preferable thatlower eukaryote host cells such as yeast and filamentous fungi begenetically engineered to include the above transporters.

Host cells further include lower eukaryote cells (e.g., yeast such asPichia pastoris) that are genetically engineered to eliminateglycoproteins having α-mannosidase-resistant N-glycans by deleting ordisrupting one or more of the β-mannosyltransferase genes (e.g., BMT1,BMT2, BMT3, and BMT4)(See, U.S. Published Patent Application No.2006/0211085) and glycoproteins having phosphomannose residues bydeleting or disrupting one or both of the phosphomannosyl transferasegenes PNO1 and MNN4B (See for example, U.S. Pat. Nos. 7,198,921 and7,259,007), which in further aspects can also include deleting ordisrupting the MNN4A gene. Disruption includes disrupting the openreading frame encoding the particular enzymes or disrupting expressionof the open reading frame or abrogating translation of RNAs encoding oneor more of the β-mannosyltransferases and/or phosphomannosyltransferasesusing interfering RNA, antisense RNA, or the like. The host cells canfurther include any one of the aforementioned host cells modified toproduce particular N-glycan structures.

Host cells further include lower eukaryote cells (e.g., yeast such asPichia pastoris) that are genetically modified to controlO-glycosylation of the glycoprotein by deleting or disrupting one ormore of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr)Mannosyl Transferase genes) (PMTS) (See U.S. Pat. No. 5,714,377) orgrown in the presence of Pmtp inhibitors and/or an alpha-mannosidase asdisclosed in Published International Application No. WO 2007061631, orboth. Disruption includes disrupting the open reading frame encoding thePmtp or disrupting expression of the open reading frame or abrogatingtranslation of RNAs encoding one or more of the Pmtps using interferingRNA, antisense RNA, or the like. The host cells can further include anyone of the aforementioned host cells modified to produce particularN-glycan structures.

Pmtp inhibitors include but are not limited to a benzylidenethiazolidinediones. Examples of benzylidene thiazolidinediones that canbe used are 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid.

In particular embodiments, the function or expression of at least oneendogenous PMT gene is reduced, disrupted, or deleted. For example, inparticular embodiments the function or expression of at least oneendogenous PMT gene selected from the group consisting of the PMT1,PMT2, PMT3, and PMT4 genes is reduced, disrupted, or deleted; or thehost cells are cultivated in the presence of one or more PMT inhibitors.In further embodiments, the host cells include one or more PMT genedeletions or disruptions and the host cells are cultivated in thepresence of one or more Pmtp inhibitors. In particular aspects of theseembodiments, the host cells also express a secretedalpha-1,2-mannosidase.

PMT deletions or disruptions and/or Pmtp inhibitors controlO-glycosylation by reducing O-glycosylation occupancy, that is byreducing the total number of O-glycosylation sites on the glycoproteinthat are glycosylated. The further addition of an alpha-1,2-mannsodasethat is secreted by the cell controls O-glycosylation by reducing themannose chain length of the O-glycans that are on the glycoprotein.Thus, combining PMT deletions or disruptions and/or Pmtp inhibitors withexpression of a secreted alpha-1,2-mannosidase controls O-glycosylationby reducing occupancy and chain length. In particular circumstances, theparticular combination of PMT deletions or disruptions, Pmtp inhibitors,and alpha-1,2-mannosidase is determined empirically as particularheterologous glycoproteins (antibodies, for example) may be expressedand transported through the Golgi apparatus with different degrees ofefficiency and thus may require a particular combination of PMTdeletions or disruptions, Pmtp inhibitors, and alpha-1,2-mannosidase. Inanother aspect, genes encoding one or emore endogenousmannosyltransferase enzymes are deleted. This deletion(s) can be incombination with providing the secreted alpha-1,2-mannosidase and/or PMTinhibitors or can be in lieu of providing the secretedalpha-1,2-mannosidase and/or PMT inhibitors.

Thus, the control of O-glycosylation can be useful for producingparticular glycoproteins in the host cells disclosed herein in bettertotal yield or in yield of properly assembled glycoprotein. Thereduction or elimination of O-glycosylation appears to have a beneficialeffect on the assembly and transport of whole antibodies as theytraverse the secretory pathway and are transported to the cell surface.Thus, in cells in which O-glycosylation is controlled, the yield ofproperly assembled antibodies fragments is increased over the yieldobtained in host cells in which O-glycosylation is not controlled.

In addition, O-glycosylation may have an effect on an antibody'saffinity and/or avidity for an antigen. This can be particularlysignificant when the ultimate host cell for production of the antibodyis not the same as the host cell that was used for selecting theantibody. For example, O-glycosylation might interfere with anantibody's affinity for an antigen, thus an antibody that mightotherwise have high affinity for an antigen might not be identifiedbecause O-glycosylation may interfere with the ability of the antibodyto bind the antigen. In other cases, an antibody that has high avidityfor an antigen might not be identified because O-glycosylationinterferes with the antibody's avidity for the antigen. In the precedingtwo cases, an antibody that might be particularly effective whenproduced in a mammalian cell line might not be identified because thehost cells for identifying and selecting the antibody was of anothercell type, for example, a yeast or fungal cell (e.g., a Pichia pastorishost cell). It is well known that O-glycosylation in yeast can besignificantly different from O-glycosylation in mammalian cells. This isparticularly relevant when comparing wild type yeast O-glycosylationwith mucin-type or dystroglycan type O-glycosylation in mammals. Inparticular cases, O-glycosylation might enhance the antibody's affinityor avidity for an antigen instead of interfere. This effect isundesirable when the production host cell is to be different from thehost cell used to identify and select the antibody (for example,identification and selection is done in yeast and the production host isa mammalian cell) because in the production host the O-glycosylationwill no longer be of the type that caused the enhanced affinity oravidity for the antigen. Therefore, controlling O-glycosylation canenable use of the materials and methods herein to identify and selectantibodies with specificity for a particular antigen based upon affinityor avidity of the antibody for the antigen without identification andselection of the antibody being influenced by the O-glycosylation systemof the host cell. Thus, controlling O-glycosylation further enhances theusefulness of yeast or fungal host cells to identify and selectantibodies that will ultimately be produced in a mammalian cell line.

Yield of antibodies can in some situations be improved by overexpressingnucleic acid molecules encoding mammalian or human chaperone proteins orreplacing the genes encoding one or more endogenous chaperone proteinswith nucleic acid molecules encoding one or more mammalian or humanchaperone proteins. In addition, the expression of mammalian or humanchaperone proteins in the host cell also appears to controlO-glycosylation in the cell. Thus, further included are the host cellsherein wherein the function of at least one endogenous gene encoding achaperone protein has been reduced or eliminated, and a vector encodingat least one mammalian or human homolog of the chaperone protein isexpressed in the host cell. Also included are host cells in which theendogenous host cell chaperones and the mammalian or human chaperoneproteins are expressed. In further aspects, the lower eukaryotic hostcell is a yeast or filamentous fungi host cell. Examples of the use ofchaperones of host cells in which human chaperone proteins areintroduced to improve the yield and reduce or control O-glycosylation ofrecombinant proteins has been disclosed in U.S. Provisional ApplicationNos. 61/066,409 filed Feb. 20, 2008 and 61/188,723 filed Aug. 12, 2008.Like above, further included are lower eukaryotic host cells wherein, inaddition to replacing the genes encoding one or more of the endogenouschaperone proteins with nucleic acid molecules encoding one or moremammalian or human chaperone proteins or overexpressing one or moremammalian or human chaperone proteins as described above, the functionor expression of at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein is reduced, disrupted, or deleted.In particular embodiments, the function of at least one endogenous PMTgene selected from the group consisting of the PMT1, PMT2, PMT3, andPMT4 genes is reduced, disrupted, or deleted.

Therefore, the methods disclose herein can use any host cell that hasbeen genetically modified to produce glycoproteins that have noN-glycans compositions wherein the predominant N-glycan is selected fromthe group consisting of complex N-glycans, hybrid N-glycans, and highmannose N-glycans wherein complex N-glycans are selected from the groupconsisting of Man₃GlcNAc₂, GlcNAC₍₁₋₄₎Man₃GlcNAc₂,Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂, and NANA₍₁₋₄₎Gal₍₁₋₄₎Man₃GlcNAc₂; hybridN-glycans are selected from the group consisting of Man₅GlcNAc₂,GlcNAcMan₅GlcNAc₂, GalGlcNAcMan₅GlcNAc₂, and NANAGalGlcNAcMan₅GlcNAc₂;and high Mannose N-glycans are selected from the group consisting ofMan₆GlcNAc₂, Man₇GlcNAc₂, Man₈GlcNAc₂, and Man₉GlcNAc₂. In particularaspects, the composition of N-glycans comprises about 39%GlcNAC₂Man₃GlcNAc₂; 40% Gal₁GlcNAC₂Man₃GlcNAc₂; and 6%Gal₂GlcNAC₂Man₃GlcNAc₂ or about 60% GlcNAC₂Man₃GlcNAc₂; 17%Gal₁GlcNAC₂Man₃GlcNAc₂; and 5% Gal₂GlcNAC₂Man₃GlcNAc₂, or mixtures inbetween.

In the above embodiments in which the yeast cell does not display1,6-mannosyl transferase activity (that is, the OCH1 gene encoding och1phas been disrupted or deleted), the host cell is not capable of mating.Thus, depending on the efficiency of transformation, the potentiallibrary diversity of light chains and heavy chains appears to be limitedto a heavy chain library of between about 10³ to 10⁶ diversity and alight chain library of about 10³ to 10⁶ diversity. However, in a yeasthost cell that is capable of mating, the diversity can be increased toabout 10⁶ to 10¹² because the host cells expressing the heavy chainlibrary can be mated to host cells expressing the light chain library toproduce host cells that express heavy chain/light chain library.Therefore, in particular embodiments, the host cell is a yeast cell suchas Pichia pastoris that displays 1,6-mannosyl transferase activities(that is, has an OCH1 gene encoding a function och1p) but which ismodified as described herein to display antibodies or fragments thereofon the cell surface. In these embodiments, the host cell can be a hostcell with its native glycosylation pathway.

Yeast selectable markers that can be used in the present inventioninclude drug resistance markers and genetic functions which allow theyeast host cell to synthesize essential cellular nutrients, e.g. aminoacids. Drug resistance markers which are commonly used in yeast includechloramphenicol, kanamycin, methotrexate, G418 (geneticin), Zeocin, andthe like. Genetic functions which allow the yeast host cell tosynthesize essential cellular nutrients are used with available yeaststrains having auxotrophic mutations in the corresponding genomicfunction. Common yeast selectable markers provide genetic functions forsynthesizing leucine (LEU2), tryptophan (TRP1 and TRP2), proline (PRO1),uracil (URA3, URA5, URA6), histidine (HIS3), lysine (LYS2), adenine(ADE1 or ADE2), and the like. Other yeast selectable markers include theARR3 gene from S. cerevisiae, which confers arsenite resistance to yeastcells that are grown in the presence of arsenite (Bobrowicz et al.,Yeast, 13:819-828 (1997); Wysocki et al., J. Biol. Chem. 272:30061-30066(1997)). A number of suitable integration sites include those enumeratedin U.S. Published application No. 2007/0072262 and include homologs toloci known for Saccharomyces cerevisiae and other yeast or fungi.Methods for integrating vectors into yeast are well known, for example,see U.S. Pat. No. 7,479,389, WO2007136865, and PCT/US2008/13719.Examples of insertion sites include, but are not limited to, Pichia ADEgenes; Pichia TRP (including TRP1 through TRP2) genes; Pichia MCA genes;Pichia CYM genes; Pichia PEP genes; Pichia PRB genes; and Pichia LEUgenes. The Pichia ADE1 and ARG4 genes have been described in LinCereghino et al., Gene 263:159-169 (2001) and U.S. Pat. No. 4,818,700,the HIS3 and TRP1 genes have been described in Cosano et al., Yeast14:861-867 (1998), HIS4 has been described in GenBank Accession No.X56180.

In embodiments that express whole antibodies, the nucleic acid moleculeencoding the antibody or heavy chain fragment thereof is modified toreplace the codon encoding an asparagine residue at position 297 of themolecule (the glycosylation site) with a codon encoding any other aminoacid residue. Thus, the antibody that is produced in the host cell isnot glycosylated. In this embodiment, the host cell displaying the heavychain library is mated to the host cell displaying the light chainlibrary and the resulting combinatorial library is screened as taughtherein. Because the antibodies lack N-glycosylation, the non-human yeastN-glycans of the host cell which might interfere with antibody affinityfor a desired antigen are not present on the recombinant antibodies.Cells producing antibodies that have desired affinity for an antigen ofinterest are selected. The nucleic acid molecules encoding the heavy andlight chains of the antibody thereof are removed from the cells and thenucleic acid molecule encoding the heavy chain is modified toreintroduce an asparagine residue at position 297. This enablesappropriate human-like glycosylation at position 297 of the antibody orfragment thereof when the nucleic acid molecule encoding the antibodythereof is introduced into a host cell that has been engineered to makeglycoproteins that have hybrid or complex N-glycans as discussedpreviously.

The cell systems used for recombinant expression and display of theimmunoglobulin can also be any higher eukaryote cell, tissue, organismfrom the animal kingdom, for example transgenic goats, transgenicrabbits, CHO cells, insect cells, and human cell lines. Examples ofanimal cells include, but are not limited to, SC-I cells, LLC-MK cells,CV-I cells, CHO cells, COS cells, murine cells, human cells, HeLa cells,293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells,RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5cells, T-FLY cells, BHK cells, SP2/0, NSO cells, and derivativesthereof. Insect cells include cells of Drosophila melanogaster origin.These cells can be genetically engineered to render the cells capable ofmaking immunoglobulins that have particular or predominantly particularN-glycans. For example, U.S. Pat. No. 6,949,372 discloses methods formaking glycoproteins in insect cells that are sialylated. Yamane-Ohnukiet al. Biotechnol. Bioeng. 87: 614-622 (2004), Kanda et al., Biotechnol.Bioeng. 94: 680-688 (2006), Kanda et al., Glycobiol. 17: 104-118 (2006),and U.S. Pub. Application Nos. 2005/0216958 and 2007/0020260 disclosemammalian cells that are capable of producing immunoglobulins in whichthe N-glycans thereon lack fucose or have reduced fucose.

In particular embodiments, the higher eukaryote cell, tissue, organismcan also be from the plant kingdom, for example, wheat, rice, corn,tobacco, and the like. Alternatively, bryophyte cells can be selected,for example from species of the genera Physcomitrella, Funaria,Sphagnum, Ceratodon, Marchantia, and Sphaerocarpos. Exemplary of plantcells is the bryophyte cell of Physcomitrella patens, which has beendisclosed in WO 2004/057002 and WO2008/006554. Expression systems usingplant cells can further manipulated to have altered glycosylationpathways to enable the cells to produce immunoglobulins that havepredominantly particular N-glycans. For example, the cells can begenetically engineered to have a dysfunctional or no corefucosyltransferase and/or a dysfunctional or no xylosyltransferase,and/or a dysfunctional or no β1,4-galactosyltransferase. Alternatively,the galactose, fucose and/or xylose can be removed from theimmunoglobulin by treatment with enzymes removing the residues. Anyenzyme resulting in the release of galactose, fucose and/or xyloseresidues from N-glycans which are known in the art can be used, forexample α-galactosidase, β-xylosidase, and α-fucosidase. Alternativelyan expression system can be used which synthesizes modified N-glycanswhich can not be used as substrates by 1,3-fucosyltransferase and/or1,2-xylosyltransferase, and/or 1,4-galactosyltransferase. Methods formodifying glycosylation pathways in plant cells has been disclosed inU.S. Published Application No. 2004/0018590.

The methods disclosed herein can be adapted for use in mammalian,insect, and plant cells. The regulatable promoters selected forregulating expression of the expression cassettes in mammalian, insect,or plant cells should be selected for functionality in the cell-typechosen. Examples of suitable regulatable promoters include but are notlimited to the tetracycline-regulatable promoters (See for example,Berens & Hillen, Eur. J. Biochem. 270: 3109-3121 (2003)), RU486-inducible promoters, ecdysone-inducible promoters, andkanamycin-regulatable systems. These promoters can replace the promotersexemplified in the expression cassettes described in the examples. Thecapture moiety can be fused to a cell surface anchoring protein suitablefor use in the cell-type chosen. Cell surface anchoring proteinsincluding GPI proteins are well known for mammalian, insect, and plantcells. GPI-anchored fusion proteins has been described by Kennard etal., Methods Biotechnol. Vo. 8: Animal Cell Biotechnology (Ed. JenkinsHuman Press, Inc., Totowa, N.J.) pp. 187-200 (1999). The genometargeting sequences for integrating the expression cassettes into thehost cell genome for making stable recombinants can replace the genometargeting and integration sequences exemplified in the examples.Transfection methods for making stable and transiently transfectedmammalian, insect, plant host cells are well known in the art. Once thetransfected host cells have been constructed as disclosed herein, thecells can be screened for expression of the immunoglobulin of interestand selected as disclosed herein.

The present invention also encompasses kits containing the expressionand helper vectors of this invention in suitable packaging. Each kitnecessarily comprises the reagents which render the delivery of vectorsinto a host cell possible. The selection of reagents that facilitatedelivery of the vectors may vary depending on the particulartransfection or infection method used. The kits may also containreagents useful for generating labeled polynucleotide probes orproteinaceous probes for detection of exogenous sequences and theprotein product. Each reagent can be supplied in a solid form ordissolved/suspended in a liquid buffer suitable for inventory storage,and later for exchange or addition into the reaction medium when theexperiment is performed. Suitable packaging is provided. The kit canoptionally provide additional components that are useful in theprocedure. These optional components include, but are not limited to,buffers, capture reagents, developing reagents, labels, reactingsurfaces, means for detection, control samples, instructions, andinterpretive information.

All publications, patents, and other references mentioned herein arehereby incorporated by reference in their entireties.

The following examples are intended to promote a further understandingof the present invention.

Example 1

Utility of the invention was demonstrated using Pichia pastoris as amodel. The glycoengineered Pichia pastoris strain yGLY2696 was thebackground strain used. In strain yGLY2696, the gene encoding theendogenous PDI replaced with a nucleic acid molecule encoding the humanPDI and a nucleic acid molecule encoding the human GRP94 proteininserted into the PEP4 locus. The strain was further engineered to alterthe endogenous glycosylation pathway to produce glycoproteins that havepredominantly Man₅GlcNAc₂ N-glycans. Strain YGLY2696 has been disclosedin co-pending Application Ser. No. 61/066,409, filed 20 Feb. 2008. Thisstrain was shown to be useful for producing immunoglobulins and forproducing immunoglobulins that have reduced O-glycosylation.Construction of strain yGLY2696 involved the following steps.

Construction of expression/integration plasmid vector pGLY642 comprisingan expression cassette encoding the human PDI protein and nucleic acidmolecules to target the plasmid vector to the Pichia pastoris PDI1 locusfor replacement of the gene encoding the Pichia pastoris PDI1 with anucleic acid molecule encoding the human PDI was as follows and is shownin FIG. 8. cDNA encoding the human PDI1 was amplified by PCR using theprimers hPDI/UP1: 5′ AGCGC TGACG CCCCC GAGGA GGAGG ACCAC 3′ (SEQ IDNO: 1) and hPDI/LP-PacI: 5′ CCTTA ATTAA TTACA GTTCA TCATG CACAG CTTTCTGATC AT 3′ (SEQ ID NO: 2), Pfu turbo DNA polymerase (Stratagene, LaJolla, Calif.), and a human liver cDNA (BD Bioscience, San Jose,Calif.). The PCR conditions were 1 cycle of 95° C. for two minutes, 25cycles of 95° C. for 20 seconds, 58° C. for 30 seconds, and 72° C. for1.5 minutes, and followed by one cycle of 72° C. for 10 minutes. Theresulting PCR product was cloned into plasmid vector pCR2.1 to makeplasmid vector pGLY618. The nucleotide and amino acid sequences of thehuman PDI1 (SEQ ID NOs:39 and 40, respectively) are shown in Table 1.

The nucleotide and amino acid sequences of the Pichia pastoris PDI1 (SEQID NOs:41 and 42, respectively) are shown in Table 1. Isolation ofnucleic acid molecules comprising the Pichia pastoris PDI1 5′ and 3′regions was performed by PCR amplification of the regions from Pichiapastoris genomic DNA. The 5′ region was amplified using primers PB248:5′ ATGAA TTCAG GCCAT ATCGG CCATT GTTTA CTGTG CGCCC ACAGT AG 3′ (SEQ IDNO: 3); PB249: 5′ ATGTT TAAAC GTGAG GATTA CTGGT GATGA AAGAC 3′ (SEQ IDNO: 4). The 3′ region was amplified using primers PB250: 5′ AGACT AGTCTATTTG GAGAC ATTGA CGGAT CCAC 3′ (SEQ ID NO: 5); PB251: 5′ ATCTC GAGAGGCCAT GCAGG CCAAC CACAA GATGA ATCAA ATTTT G-3′ (SEQ ID NO: 6). Pichiapastoris strain NRRL-11430 genomic DNA was used for PCR amplification.The PCR conditions were one cycle of 95° C. for two minutes, 25 cyclesof 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2.5minutes, and followed by one cycle of 72° C. for 10 minutes. Theresulting PCR fragments, PpPDI1 (5′) and PpPDI1 (3′), were separatelycloned into plasmid vector pCR2.1 to make plasmid vectors pGLY620 andpGLY617, respectively. To construct pGLY678, DNA fragments PpARG3-5′ andPpARG-3′ of integration plasmid vector pGLY24, which targets the plasmidvector to Pichia pastoris ARG3 locus, were replaced with DNA fragmentsPpPDI (5′) and PpPDI (3′), respectively, which targets the plasmidvector pGLY678 to the PDI1 locus and disrupts expression of the PDI1locus.

The nucleic acid molecule encoding the human PDI was then cloned intoplasmid vector pGLY678 to produce plasmid vector pGLY642 in which thenucleic acid molecule encoding the human PDI was placed under thecontrol of the Pichia pastoris GAPDH promoter (PpGAPDH).Expression/integration plasmid vector pGLY642 was constructed byligating a nucleic acid molecule encoding the Saccharomyces cerevisiaealpha mating factor (MF) presequence signal peptide (ScaMFpre-signalpeptide) having a NotI restriction enzyme site at the 5′ end and a blunt3′ end and the expression cassette comprising the nucleic acid moleculeencoding the human PDI released from plasmid vector pGLY618 with Afeland Pad to produce a nucleic acid molecule having a blunt 5′ end and aPad site at the 3′ end into plasmid vector pGLY678 digested with NotIand Pad. The resulting integration/expression plasmid vector pGLY642comprises an expression cassette encoding a human PDI1/ScaMFpre-signalpeptide fusion protein operably linked to the Pichia pastoris promoterand nucleic acid molecule sequences to target the plasmid vector to thePichia pastoris PDI1 locus for disruption of the PDI1 locus andintegration of the expression cassette into the PDI1 locus. FIG. 2illustrates the construction of plasmid vector pGLY642. The nucleotideand amino acid sequences of the ScαMFpre-signal peptide are shown in SEQID NOs:27 and 28, respectively.

Construction of expression/integration vector pGLY2233 encoding thehuman GRP94 protein was as follows and is shown in FIG. 3. The humanGRP94 was PCR amplified from human liver cDNA (BD Bioscience) with theprimers hGRP94/UP1: 5′-AGCGC TGACG ATGAA GTTGA TGTGG ATGGT ACAGT AG-3;(SEQ ID NO: 15); and hGRP94/LP1: 5′-GGCCG GCCTT ACAAT TCATC ATGTT CAGCTGTAGA TTC 3′; (SEQ ID NO: 16). The PCR conditions were one cycle of 95°C. for two minutes, 25 cycles of 95° C. for 20 seconds, 55° C. for 20seconds, and 72° C. for 2.5 minutes, and followed by one cycle of 72° C.for 10 minutes. The PCR product was cloned into plasmid vector pCR2.1 tomake plasmid vector pGLY2216. The nucleotide and amino acid sequences ofthe human GRP94 (SEQ ID NOs:43 and 44, respectively) are shown in Table1.

The nucleic acid molecule encoding the human GRP94 was released fromplasmid vector pGLY2216 with Afel and FseI. The nucleic acid moleculewas then ligated to a nucleic acid molecule encoding the ScαMPpre-signalpeptide having NotI and blunt ends as above and plasmid vector pGLY2231digested with NotI and FseI carrying nucleic acid molecules comprisingthe Pichia pastoris PEP4 5′ and 3′ regions (PpPEP4-5′ and PpPEP4-3′regions, respectively) to make plasmid vector pGLY2229. Plasmid vectorpGLY2229 was digested with BglII and NotI and a DNA fragment containingthe PpPDI1 promoter was removed from plasmid vector pGLY2187 with Bg/IIand NotI and the DNA fragment ligated into pGLY2229 to make plasmidvector pGLY2233. Plasmid vector pGLY2233 encodes the human GRP94 fusionprotein under control of the Pichia pastoris PDI promoter and includesthe 5′ and 3′ regions of the Pichia pastoris PEP4 gene to target theplasmid vector to the PEP4 locus of genome for disruption of the PEP4locus and integration of the expression cassette into the PEP4 locus.FIG. 3 illustrates the construction of plasmid vector pGLY2233.

Construction of plasmid vectors pGLY1162, pGLY1896, and pGFI207t was asfollows. All Trichoderma reesei α-1,2-mannosidase expression plasmidvectors were derived from pGFI165, which encodes the T. reeseiα-1,2-mannosidase catalytic domain (See published InternationalApplication No. WO2007061631) fused to S. cerevisiae αMATpre signalpeptide (ScαMPpre-signal peptide) herein expression is under the controlof the Pichia pastoris GAP promoter and wherein integration of theplasmid vectors is targeted to the Pichia pastoris PRO1 locus andselection is using the Pichia pastoris URA5 gene. A map of plasmidvector pGFI165 is shown in FIG. 4.

Plasmid vector pGLY1162 was made by replacing the GAP promoter inpGFI165 with the Pichia pastoris AOX1 (PpAOX1) promoter. This wasaccomplished by isolating the PpAOX1 promoter as an EcoRI (madeblunt)-BglII fragment from pGLY2028, and inserting into pGFI165 that wasdigested with Nod (made blunt) and BglII. Integration of the plasmidvector is to the Pichia pastoris PRO1 locus and selection is using thePichia pastoris URA5 gene. A map of plasmid vector pGLY1162 is shown inFIG. 5.

Plasmid vector pGLY1896 contains an expression cassette encoding themouse α-1,2-mannosidase catalytic domain fused to the S. cerevisiae MNN2membrane insertion leader peptide fusion protein (See Choi et al., Proc.Natl. Acad. Sci. USA 100: 5022 (2003)) inserted into plasmid vectorpGFI165 (FIG. 5). This was accomplished by isolating theGAPp-ScMNN2-mouse MNSI expression cassette from pGLY1433 digested withXhoI (and the ends made blunt) and PmeI, and inserting the fragment intopGFI165 that digested with PmeI. Integration of the plasmid vector is tothe Pichia pastoris PRO1 locus and selection is using the Pichiapastoris URA5 gene. A map of plasmid vector pGLY1896 is shown in FIG. 4.

Plasmid vector pGFI207t is similar to pGLY1896 except that the URA5selection marker was replaced with the S. cerevisiae ARR3 (ScARR3) gene,which confers resistance to arsenite. This was accomplished by isolatingthe ScARR3 gene from pGFI166 digested with AscI and the AscI ends madeblunt) and Bg/II, and inserting the fragment into pGLY1896 that digestedwith SpeI and the SpeI ends made blunt and Bg/II. Integration of theplasmid vector is to the Pichia pastoris PRO1 locus and selection isusing the Saccharomyces cerevisiae ARR3 gene. A map of plasmid vectorpGFI2007t is shown in FIG. 4. The ARR3 gene from S. cerevisiae confersarsenite resistance to cells that are grown in the presence of arsenite(Bobrowicz et al., Yeast, 13:819-828 (1997); Wysocki et al., J. Biol.Chem. 272:30061-066 (1997)).

Yeast transfections with the above expression/integration vectors wereas follows. Pichia pastoris strains were grown in 50 mL YPD media (yeastextract (1%), peptone (2%), dextrose (2%)) overnight to an OD of betweenabout 0.2 to 6. After incubation on ice for 30 minutes, cells werepelleted by centrifugation at 2500-3000 rpm for 5 minutes. Media wasremoved and the cells washed three times with ice cold sterile 1Msorbitol before resuspending in 0.5 ml ice cold sterile 1M sorbitol. TenμL linearized DNA (5-20 μg) and 100 4 cell suspension was combined in anelectroporation cuvette and incubated for 5 minutes on ice.Electroporation was in a Bio-Rad GenePulser Xcell following the presetPichia pastoris protocol (2 kV, 25 μF, 200Ω), immediately followed bythe addition of 1 mL YPDS recovery media (YPD media plus 1M sorbitol).The transfected cells were allowed to recover for four hours toovernight at room temperature (26° C.) before plating the cells onselective media.

Generation of Cell Lines was as follows and is shown in FIG. 6. Thestrain yGLY24-1 (ura5Δ::MET1 och1 Δ::lacZbmt2Δ::lacZ/KlMNN2-2/mnn4L1Δ::lacZ/MmSLC35A3 pnolΔmnn4Δ::lacZmetl6Δ::lacZ), was constructed using methods described earlier (See forexample, Nett and Gerngross, Yeast 20:1279 (2003); Choi et al., Proc.Natl. Acad. Sci. USA 100:5022 (2003); Hamilton et al., Science 301:1244(2003)). The BMT2 gene has been disclosed in Mille et al., J. Biol.Chem. 283: 9724-9736 (2008) and U.S. Published Application No.20060211085. The PNO1 gene has been disclosed in U.S. Pat. No. 7,198,921and the mnn4L1 gene (also referred to as mnn4b) has been disclosed inU.S. Pat. No. 7,259,007. The mnn4 refers to mnn4L2 or mnn4a. In thegenotype, KlMNN2-2 is the Kluveromyces lactis GlcNAc transporter andMmSLC35A3 is the Mus musculus GlcNAc transporter. The URA5 deletionrenders the yGLY24-1 strain auxotrophic for uracil (See U.S. Publishedapplication No. 2004/0229306) and was used to construct the humanizedchaperone strains that follow. While the various expression cassetteswere integrated into particular loci of the Pichia pastoris genome inthe examples herein, it is understood that the operation of theinvention is independent of the loci used for integration. Loci otherthan those disclosed herein can be used for integration of theexpression cassettes. Suitable integration sites include thoseenumerated in U.S. Published application No. 20070072262 and includehomologs to loci known for Saccharomyces cerevisiae and other yeast orfungi.

Strains yGLY702 and yGLY704 were generated in order to test theeffectiveness of the human PDI1 expressed in Pichia pastoris cells inthe absence of the endogenous Pichia pastoris PDI gene. Strains yGLY702and yGLY704 (huPDI) were constructed as follows. Strain yGLY702 wasgenerated by transfecting yGLY24-1 with plasmid vector pGLY642containing the expression cassette encoding the human PDI under controlof the constitutive PpGAPDH promoter. Plasmid vector pGLY642 alsocontained an expression cassette encoding the Pichia pastoris URA5,which rendered strain yGLY702 prototrophic for uracil. The URA5expression cassette was removed by counterselecting yGLY702 on 5-FOAplates to produce strain yGLY704 in which, so that the Pichia pastorisPDI1 gene has been stably replaced by the human PDI gene and the strainis auxotrophic for uracil.

Strain yGLY733 was generated by transfecting with plasmid vectorpGLY1162, which comprises an expression cassette that encodes theTrichoderma Reesei mannosidase (TrMNS1) operably linked to the Pichiapastoris AOX1 promoter (PpAOX1-TrMNS1), into the PRO1 locus of yGLY704.This strain has the gene encoding the Pichia pastoris PD1 replaced withthe expression cassette encoding the human PDI1, has the PpAOX1-TrMNS1expression cassette integrated into the PRO1 locus, and is a URA5auxotroph. The PpAOX1 promoter allows overexpression when the cells aregrown in the presence of methanol.

Strain yGLY762 was constructed by integrating expression cassettesencoding TrMNS1 and mouse mannosidase IA (MuMNS1A), each operably linkedto the Pichia pastoris GAPDH promoter in plasmid vector pGFI207t intocontrol strain yGLY733 at the 5′ PRO1 locus UTR in Pichia pastorisgenome. This strain has the gene encoding the Pichia pastoris PD1replaced with the expression cassette encoding the human PDI1, has thePpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated intothe PRO1 locus, and is a URA5 auxotroph.

Strain yGLY2677 was generated by counterselecting yGLY762 on 5-FOAplates. This strain has the gene encoding the Pichia pastoris PD1replaced with the expression cassette encoding the human PDI1, has thePpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, hasthe PpGAPH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integratedinto the PRO1 locus, and is a URA5 prototroph.

Strains yGLY2696 was generated by integrating plasmid vector pGLY2233,which encodes the human GRP94 protein, into the PEP4 locus. This strainhas the gene encoding the Pichia pastoris PD1 replaced with theexpression cassette encoding the human PDI1, has the PpAOX1-TrMNS1expression cassette integrated into the PRO1 locus, has thePpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated intothe PRO1 locus, has the human GRP64 integrated into the PEP4 locus, andis a URA5 prototroph. The genealogy of this chaperone-humanized strainis shown in FIG. 6.

Example 2

Expression vectors encoding an anti-Her2 antibody and an anti-CD20antibody were constructed as follows.

Expression/integration plasmid vector pGLY2988 contains expressioncassettes encoding the heavy and light chains of an anti-Her2 antibody.Anti-Her2 heavy (HC) and light (LC) chains fused at the N-terminus toα-MAT pre signal peptide were synthesized by GeneArt AG. The nucleotideand amino acid sequences for the α-amylase signal peptide are shown inSEQ ID NOs:27 and 28. Each was synthesized with unique 5′ EcoR1 and 3′Fse1 sites. The nucleotide and amino acid sequences of the anti-Her2 HCare shown in SEQ ID Nos:29 and 30, respectively. The nucleotide andamino acid sequences of the anti-Her2 LC are shown in SEQ ID Nos:31 and32, respectively. Both nucleic acid molecule fragments encoding the HCand LC fusion proteins were separately subcloned using 5′ EcoR1 and 3′Fse1 unique sites into an expression plasmid vector pGLY2198 (containsthe Pichia pastoris TRP2 targeting nucleic acid molecule and theZeocin-resistance marker) to form plasmid vector pGLY2987 and pGLY2338,respectively. The LC expression cassette encoding the LC fusion proteinunder the control of the Pichia pastoris AOX1 promoter and Saccharomycescerevisiae CYC terminator was removed from plasmid vector pGLY2338 bydigesting with BamH1 and NotI and then cloning the DNA fragment intoplasmid vector pGLY2987 digested with BamH1 and Not1, thus generatingthe final expression plasmid vector pGLY2988 (FIG. 7).

Expression/integration plasmid vector pGLY3200 (map is identical topGLY2988 except LC and HC are anti-CD20 with α-amylase signalsequences). Anti-CD20 sequences were from GenMab sequence 2C6 exceptLight chain (LC) framework sequences matched those from VKappa 3germline. Heavy (HC) and LC variable sequences fused at the N-terminusto the α-amylase (from Aspergillus niger) signal peptide weresynthesized by GeneArt AG. The nucleotide and amino acid sequences forthe α-amylase signal peptide are shown in SEQ ID NOs:33 and 34. Each wassynthesized with unique 5′ EcoR1 and 3′ KpnI sites which allowed for thedirect cloning of variable regions into expression vectors containingthe IgG1 and V kappa constant regions. The nucleotide and amino acidsequences of the anti-CD20 HC are shown in SEQ ID Nos:37 and 38,respectively. The nucleotide and amino acid sequences of the anti-CD LCare shown in SEQ ID Nos:35 and 36, respectively. Both HC and LC fusionproteins were subcloned into IgG1 plasmid vector pGLY3184 and VKappaplasmid vector pGLY2600, respectively, (each plasmid vector contains thePichia pastoris TRP2 targeting nucleic acid molecule andZeocin-resistance marker) to form plasmid vectors pGLY3192 and pGLY3196,respectively. The LC expression cassette encoding the LC fusion proteinunder the control of the Pichia pastoris AOX1 promoter and Saccharomycescerevisiae CYC terminator was removed from plasmid vector pGLY3196 bydigesting with BamHI and NotI and then cloning the DNA fragment intoplasmid vector pGLY3192 digested with BamH1 and Not1, thus generatingthe final expression plasmid vector pGLY3200 (FIG. 8).

Transfection of strain yGLY2696 with the above anti-Her2 or anti-CD20antibody expression/integration plasmid vectors was performedessentially as follows. Appropriate Pichia pastoris strains were grownin 50 mL YPD media (yeast extract (1%), peptone (2%), dextrose (2%))overnight to an OD of between about 0.2 to 6. After incubation on icefor 30 minutes, cells were pelleted by centrifugation at 2500-3000 rpmfor 5 minutes. Media were removed and the cells washed three times withice cold sterile 1M sorbitol before resuspending in 0.5 mL ice coldsterile 1M sorbitol. Ten μL linearized DNA (5-20 μg) and 100 μL cellsuspension was combined in an electroporation cuvette and incubated for5 minutes on ice. Electroporation was in a Bio-Rad GenePulser Xcellfollowing the preset Pichia pastoris protocol (2 kV, 25 μF, 200Ω),immediately followed by the addition of 1 mL YPDS recovery media (YPDmedia plus 1M sorbitol). The transfected cells were allowed to recoverfor four hours to overnight at room temperature (26° C.) before platingthe cells on selective media. Strain yGLY2696 transfected with pGLY2988encoding the anti-HER2 antibody was designated yGLY4134. Strain yGLY2696transfected with pGLY3200 encoding the anti-CD20 antibody was designatedyGLY3920.

Example 3

This example describes the construction of plasmids comprisingexpression cassettes encoding cell surface anchoring proteins fused tobinding moieties capable of binding an immunoglobulin, which aresuitable for use in Pichia pastoris. The plasmids comprise a nucleicacid molecule encoding sed1p, a cell surface anchoring protein thatinherently contains an attached glycophosphotidylinositol (GPI)post-translational modification that anchors the protein in the cellwall. The nucleic acid molecule encoding the sed1p was linked in frameto a nucleic acid molecule encoding an antibody-binding moiety that iscapable of binding whole, intact antibodies.

Four plasmids were constructed containing antibody binding moiety/cellsurface anchor fusion protein expression cassettes. Plasmid pGLY4136encodes the five Fc binding domains of Protein A fused to theSaccharomyces cerevisiae SED1 (ScSED1) gene followed by the CYCterminator, all under the control of the AOX promoter (FIG. 9). PlasmidpGLY4116 encodes the Fc receptor III (FcRIII (LF)) fused to the ScSED1gene (FIG. 10). Plasmid pGLY4137 encodes Fc receptor I (FcRI) fused tothe ScSED1 gene (FIG. 10) and plasmid pGLY4124 (FIG. 9) encodes theZZ-domain from Protein A fused to the ScSED1 gene. The ZZ-domainconsists of two of the five Fc binding domains. All four plasmidscontain a pUC19 E. coli origin and an arsenite resistance marker and areintegrated into the Pichia pastoris genome at the URA6 locus.

Plasmid pGLY3033 comprising an expression cassette encoding a fusionprotein comprising the Saccharomyces cerevisiae SED1 GPI anchoringprotein without its endogenous signal peptide (SER1 fragment) has beendescribed in copending Application Ser. No. 61/067,965 filed Mar. 3,2008. The SED1 amino acid sequence without its endogenous signal peptideis shown in SEQ ID NO:60. A nucleic acid molecule encoding the SED1fragment was synthesized by GeneArt AG. The codons encoding the fragmenthad been optimized for expression in Pichia pastoris. The nucleotidesequence encoding the SED1 fragment is shown in SEQ ID NO:61). ThePichia pastoris URA6 locus was chosen as an integrating site for the GPIanchoring protein expression cassette. The URA6 gene was PCR amplifiedfrom Pichia pastoris genomic DNA and cloned into pCR2.1 TOPO(Invitrogen, La Jolla, Calif.) to produce plasmid pGLY1849. The Bg/IIand EcoRI sites within the gene were mutated by silent mutation forcloning purposes. The TRP2 targeting nucleic acid molecule of plasmidpGLY2184 was replaced with the Pichia pastoris URA6 gene from pGLY1849.In addition, the Pichia pastoris ARM selection marker was replaced withthe Arsenite marker cassette from plasmid pGFI8. The final plasmid wasnamed pGFI30t and was used to make plasmid pGLY3033 (FIG. 20),containing an expression cassette comprising a nucleic acid moleculeencoding the SED1 fragment protein fused at its amino terminus to a GR2coiled-coil peptide and Aspergillus niger alpha-amylase signal peptideoperably linked to the PpAOX1 promoter. The GR2 coiled coil and signalpeptide encoding fragment can be removed by EcoRI and SalI digestion andreplaced with an antibody capture moiety to make a fusion protein inwhich the capture moiety is fused to a cell surface anchoring protein.

Plasmid pGLY4136 comprising an expression cassette encoding the five Fcbinding domains of protein A fused to the SED1 fragment under thecontrol of the AOX1 promoter was constructed as follows. A nucleic acidmolecule fragment encoding the five Fc binding domains from protein Awas synthesized by GeneArt to encode the five Fc binding domains fusedto the Saccharomyces cerevisiae α-Mating Factor pre signal sequence atthe N-terminus and an HA and 9×HIS Tag sequence at the C-terminus and tohave an EcoRI 5′ end and a SalI3′ end. The fragment apre-5xBD-Htag hasthe nucleotide sequence shown in SEQ ID NO:45. The apre-5xBD-Htag fusionprotein has the amino acid sequence shown in SEQ ID NO:46. The nucleicacid molecule encoding the apre-5xBD-Htag fusion protein was digestedwith EcoRI and SalI and the fragment cloned into pGLY3033, which hadbeen digested with EcoRI and SalI to remove the GR2 coiled coil encodingfragment. This produced plasmid pGLY4136, which contains operably linkedto the PpAOX1 promoter, the nucleic acid molecule encoding theapre-5xBD-Htag fusion protein linked in-frame to the nucleic acidmolecule encoding the SED1 fragment. The plasmid is anintegration/expression vector that targets the plasmid to the URA6locus. The fusion protein expressed by this integration/insertionplasmid is referred to herein as the Protein A/SED1 fusion protein.

To put the Protein A/SED1 fusion protein under the control of the GAPDHpromoter, plasmid pGLY4136 was digested with Bg/II and EcoRI to releasethe AOX1 promoter and to insert the Pichia pastoris GAPDH promoter frompGLY880. This produced plasmid pGLY4139.

Plasmid pGLY4124 comprising an expression cassette encoding the ProteinA ZZ domain fused to the SED1 fragment under the control of the AOX1promoter was constructed as follows. The ZZ-domain from GeneArt plasmid0706208 ZZHAtag was PCR amplified using the following primers: primeralpha-amy-ProtAZZ/up:CGGAATTCacgATGGTCGCTTGGTGGTCTTTGTTTCTGTACGGTCTTCAGGTCGCTGCACCTGCTTTGGCTTCTGGTGGTGTTACTCCAGCTGCTAACGCTGCTCAACACG (SEQ ID NO:47) andHA-ProtAZZ-XhoIZZ/lp: GCCTCGAGAGCGTAGTCTGGAACATCGTATGGGTAACCACCACCAGCATC(SEQ ID NO:48). The alpha-amy-ProtAZZ/up primer includes in-frame thecoding sequence for the first 20 amino acids of the Aspergillus nigerα-amylase signal peptide (underlined). The primers introduce an EcoRIsite at the 5′ end of the coding region and a XhoI site at the 3′ end.The nucleic acid sequence of the ZZ-domain as an EcoRI/XhoI fragment isshown in SEQ ID NO:49. The amino acid sequence of the ZZ-domain is shownin SEQ ID NO:50. The PCR conditions were one cycle of 95° C. for 2minutes, 20 cycles of 98° C. for 10 seconds, 65° C. for 10 seconds, and72° C. for 1 minute, and followed by one cycle of 72° C. for 10 minutes.

The PCR fragment was cloned into plasmid pCR2.1 TOPO and the clonedfragment sequenced to confirm the sequence encoded the Protein A ZZdomain. The ZZ-domain fragment was extracted from the pCR2.1 TOPO vectorby EcoRI and XhoI digest and the EcoRI/XhoI fragment was cloned intoplasmid pGLY3033, which had been digested with EcoRI and SalI to removethe GR2 coiled coil encoding fragment. This produced plasmid pGLY4124,which contains operably linked to the PpAOX1 promoter, the nucleic acidmolecule encoding the Protein A ZZ domain-alpha amylase signal peptidefusion protein linked in-frame to the nucleic acid molecule encoding theSER1 fragment. The plasmid is an integration/expression vector thattargets the plasmid to the URA6 locus. The fusion protein expressed bythis integration/insertion plasmid is referred to herein as the ZZ/SED1fusion protein.

Plasmid pGLY4116 comprising an expression cassette encoding the FcRIIIaLF receptor fused to the SER1 fragment under the control of the AOX1promoter was constructed as follows. A nucleic acid molecule encodingthe FcRIIIa LF receptor was PCR amplified from plasmid pGLY3247 (FcRIIIaLF) as an EcoRI/SalI fragment. In plasmid pGLY3247, the FcRIIIa LFreceptor is a fusion protein in which the endogenous signal peptide hadbeen replaced with the α-MFpre-pro. The 5′ primer anneals to thesequence encoding the signal peptide and the 3′ primer anneals to theHis-tag at the end of the receptor and omits the stop codon for thereceptor. The 5′ primer was 5Ecoapp: AACGGAATTCATGAGATTTCCTTCAATTTTTAC(SEQ ID NO:51) and the 3′ primer was 3HtagSalCGATGTCGACGTGATGGTGATGGTGGTGATGATGATGACCACC (SEQ ID NO:52). The PCRconditions were one cycle of 95° C. for 2 minutes, 25 cycles of 95° C.for 30 seconds, 58° C. for 30 seconds, and 72° C. for 70 seconds, andfollowed by one cycle of 72° C. for 10 minutes.

The PCR fragment encoding the receptor fusion protein was cloned intoplasmid pCR2.1 TOPO and the cloned fragment sequenced to confirm thesequence encoded the receptor. The nucleotide sequence of the FcRIII(LF)as an EcoRI/SalI fragment is shown in SEQ ID NO:53. The amino acidsequence of the FcRIII(LF) with a MF pre-signal sequence is shown in SEQID NO:54.

Plasmid pCR2.1 TOPO was digested with EcoRI and SalI and the EcoRI/SalIfragment encoding the receptor was cloned into pGLY3033, which had beendigested with EcoRI and SalI to remove the GR21 coiled coil encodingfragment. This produced plasmid pGLY4116, which contains operably linkedto the PpAOX1 promoter, the nucleic acid molecule encoding the FcRIIIaLF/α-MF pre-pro signal peptide fusion protein linked in-frame to thenucleic acid molecule encoding the SER1 fragment. The plasmid is anintegration/expression vector that targets the plasmid to the URA6locus. The fusion protein expressed by this integration/insertionplasmid is referred to herein as the FcRIIIa fusion protein.

Plasmid pGLY4137 encoding the FcRI receptor fused to the SER1 fragmentwas constructed as follows. A nucleic acid molecule encoding the FcRIreceptor was PCR amplified from plasmid pGLY3248 as an EcoRI/SalIfragment. In plasmid pGLY3248, the FcRI receptor is a fusion protein inwhich the endogenous signal peptide had been replaced with theα-MFpre-pro. The 5′ primer anneals to the sequence encoding the signalpeptide and the 3′ primer anneals to the His-tag at the end of thereceptor and omits the stop codon for the receptor. The 5′ primer was5Ecoapp: AACGGAATTCATGAGATTTCCTTCAATTTTTAC (SEQ ID NO:51) and the 3′primer was 3HtagSal CGATGTCGACGTGATGGTGATGGTGGTGATGATGATGACCACC (SEQ IDNO:52). The PCR conditions were one cycle of 95° C. for 2 minutes, 25cycles of 95° C. for 30 seconds, 58° C. for 30 seconds, and 72° C. for70 seconds, and followed by one cycle of 72° C. for 10 minutes.

The PCR fragment encoding the receptor fusion protein was cloned intoplasmid pCR2.1 TOPO and the cloned fragment sequenced to confirm thesequence encoded the receptor. The nucleic acid sequence of the FcRI asan EcoRI/SalI fragment is shown in SEQ ID NO:55. The amino acid sequenceof the FcRI with a MF pre-signal sequence is shown in SEQ ID NO:56.

Plasmid pCR2.1 TOPO was digested with EcoRI and SalI and the EcoRI/SalIfragment encoding the receptor was cloned into pGLY3033, which had beendigested with EcoRI and SalI to remove the GR21 coiled coil encodingfragment. This produced plasmid pGLY4116, which contains operably linkedto the PpAOX1 promoter, the nucleic acid molecule encoding the FcRI/α-MFpre-pro signal peptide fusion protein linked in-frame to the nucleicacid molecule encoding the SED1 fragment. The plasmid is anintegration/expression vector that targets the plasmid to the URA6locus. The fusion protein expressed by this integration/insertionplasmid is referred to herein as the FcRI fusion protein.

Example 4

Co-Expression of antibody and antibody binding moiety/cell surfaceanchor fusion protein in Pichia pastoris was as follows.

Pichia pastoris strains yGLY4134 (expresses anti-HER2 antibody) andyGLY3920 (expresses anti-CD20 antibody) were each transfected withpGLY4116 (expresses FcRIII receptor/SED fusion protein), pGLY4136(expresses Protein A/SED fusion protein), pGLY4124 (expresses Protein AZZ domain/SED fusion protein), or pGLY4137 (expresses FcRI receptor/SEDfusion protein). YGLY2696 was also transfected with each of the abovefour expression/integration vectors. For transfection, the strains aregrown in 50 mL BMGY media until the culture reached a density of aboutOD600=2.0. The cells are washed three times with 1M sorbitol andresuspended in 1 mL 1M sorbitol. About 1 to 2 μg of linearized plasmidare mixed with the cells. Transfection is performed with a BioRadelectroporation apparatus using the manufacturer's program specific forelectroporation of nucleic acid molecules into Pichia pastoris. One mLof recovery media is added to the cells, which are then plated out onYPG (yeast extract:peptone:glycerol medium) with 50 μg/mL arsenite.

Cell surface labeling was as follows. Strain yGLY4134 (expressesanti-Her2 antibody), strain yGLY4134 transfected with pGLY4136(expresses anti-Her2 antibody and Protein A/SED1 fusion protein, andstrain YGLY2696 transfected with pGLY4136 (expresses Protein A/SED 1fusion protein) were grown in 600 μL BMGY (buffered minimal glycerolmedium-yeast extract, Invitrogen) in a 96 deep well plate or 50 mL BMGYin a 250 mL shake flask for two days. The cells were collected bycentrifugation and the supernatant was discarded. The cells were inducedby incubation in 300 μL or 25 mL BMMY with Pmti-3 inhibitor overnightfollowing the methods taught in WO2007/061631. Pmti-3 is3-hydroxy-4-(2-phenylethoxy)benzaldehyde;3-(1-phenylethoxy)-4-(2-phenylethoxy)-benzaldehyde, which as beendescribed in U.S. Pat. No. 7,105,554 and Published InternationalApplication No. WO 2007061631. The Pmti-3 inhibitor reduces theO-glycosylation occupancy, that is the number of total O-glycans on theantibody molecule. The cell further express a T. reeseialpha-1,2-mannsodase catalytic domain linked to the Saccharomycescerevisiea αMAT pre signal peptide to control the chain length of thoseO-glycans that are on the antibody molecule.

Induced cells were labeled with goat anti-human heavy and light chain(H+L) Alexa 488 (Invitrogen, Carlsbad, Calif.) conjugated antibody andviewed using fluorescence microscopy as follows. After induction, cellsat density of about 0.5-1.0 OD600 were collected by centrifugation in a1.5-mL tube. The cells were rinsed twice with 1 mL PBS and 0.5 mL goatanti-human IgG (H+L)-Alexa 488 (1:500 in 1% BSA in PBS) was added. Thetubes were rotated for one hour at 37° C., centrifuged, and rinsed 3×with 1 mL PBS to remove the detection antibody. The cells wereresuspended in about 50-100 μL of PBS and a 10 μL aliquot viewed with afluorescence microscope and photographed (FIG. 2). As expected, both theanti-Her2 antibody expressing strain yGLY4134 without pGLY4136 encodingthe protein A/SED1 fusion protein and yGLY2696 with pGLY4136 encodingthe Protein A/SED1 fusion protein but no anti-Her2 antibody showed nosurface labeling. The weak labeling that was visible on the cells ofyGLY2696 transfected with pGLY4136 might be due to cross reaction of thegoat anti human heavy and light chain (H+L) Alexa 488 conjugatedantibody to the expressed Protein A. However, as can also be seen inFIG. 11, co-expression of the Protein A/SED1 fusion protein and theanti-Her2 antibody (strain yGLY4134 transfected with pGLY4136) did notresult in displayed antibody on the cell surface and showed onlybackground labeling. This result suggested that simultaneouslyexpressing the antibody and Protein A/SED1 protein interfered withdisplay of the antibody on the cell surface or the Protein A/SED 1protein was not properly anchored to the cell surface.

Example 5

This example demonstrates that the Protein A/SED1 fusion protein isproperly anchored to the cell surface and that co-expressing theanti-Her2 antibody and Protein A/SED1 fusion protein at the same timeinterfere with capture and display of the antibody on the cell surface.

To test whether the Protein A/SED 1 fusion protein itself is displayedon the cell surface, strain yGLY2696 transfected pGLY4136 encoding theProtein A/SED1 fusion protein was grown and induced as described in theprevious example. At a cell density of about 0.5-1.0 OD600, cells werecollected by centrifugation in a 1.5-mL tube and rinsed twice with 1 mLPBS. Either 10 or 50 ng of anti-Her2 antibody was added externally tothe cells and the cells incubated for one hour. Afterwards, the cellswere washed 3× in 1 ml PBS and labeled with goat anti human H+L asdescribed in the previous example. The results showed that the anti-Her2antibody was captured and displayed on the surface of the cells. Thiscan be seen in FIG. 12, which shows strong cell surface staining. Theresults confirm that the Protein A/SED1 fusion protein is expressed, theexpressed fusion protein is properly inserted into the cell surface, andthe fusion protein is able to capture and display antibodies on the cellsurface.

To determine whether co-expression interfered with display of theantibody on the cell surface, strain yGLY2696 transfected with pGLY4136(empty strain that expresses Protein A/SED1 fusion protein), strainyGLY4134 transfected with pGLY4136 (strain expresses anti-Her2 antibodyand Protein A/SED1 fusion protein), and strain yGLY3920 transfected withpGLY4136 (strain expresses anti-CD20 antibody and Protein A/SED1 fusionprotein) were grown and induced as in the previous example. Cells wereincubated with 10 ng externally added anti-Her2 antibody, labeled, anddetected as in the previous example. FIG. 13 illustrates strong cellsurface labeling of the empty strain expressing only the Protein A/SED 1fusion protein (yGLY2696 transfected with pGLY4136), but only weakstaining in the strains when the Protein A/SED1 fusion protein and theantibody were co-expressed (yGLY4134 transfected with pGLY4136 andyGLY3920 transfected with pGLY4136). Cells expressing the Protein A/SED1fusion protein were able to capture externally added antibody anddisplay it while cells co-expressing antibody and Protein A/SED1 fusionprotein were unable to capture externally added antibody nor displaytheir own secreted antibody.

These results suggested that the Protein A/SED1 fusion protein is notdisplayed well on the cell surface in an antibody co-expressing strain.This may be because co-expression of the Protein A/SED1 fusion proteinand the antibody from the strong AOX promoter under methanol inductionmay lead to aggregation of the antibody—Protein A/SED1 fusion proteincomplex in the ER and degradation. Alternatively, the antibody—ProteinA/SED1 fusion protein complex produced in the ER may not secrete wellbecause of its molecular weight or steric hindrance.

Example 6

Other antibody binding moieties were tested for their ability to displayantibody on the cell surface of P. pastoris. These include the Fcreceptor I (FcRI), the Fc receptor III (FcRIII) and the Protein AZZ-domain. Strains yGLY2696 (empty), yGLY4134 (expresses anti-Her2antibody) and yGLY3920 (expresses anti-CD20 antibody) were separatelytransfected with each of plasmids pGLY4116 (encodes FcRIII/SED1 fusionprotein), pGLY4124 (encodes Protein A ZZ domain/SED1 fusion protein),and pGLY4136 (encodes Protein A/SED1 fusion protein), were grown,induced and labeled as in Example 4.

The results for the ZZ-domain were similar to those for Protein A albeitthe staining was somewhat weaker. This suggests that two Fc bindingdomains have a lower affinity for the antibody compared to the intactProtein A, which has five Fc binding domains.

Co-expression of the FcRIII/SED1 fusion protein and antibody resulted ina lack of cell surface staining Strain yGLY2696 transfected withpGLY4116 (encodes FcRIII/SED1 fusion protein) was grown and induced asdescribed in Example 4 and the cells were incubated with 10 or 50 ngexternally added anti-Her2 antibody. Contrary to the results fromstrains that expressed the Protein A/SED1 fusion protein, cell surfacestaining was absent while some intracellular staining is observed (FIG.14). The results suggest that while the FcRIII/SED1 fusion protein maybe expressed in the cell, it did not appear to be secreted.

Example 7

This example demonstrates that temporal expression of the Protein A/SED1fusion protein and the antibody enables proper expression and capture ofthe secreted antibody on the cell surface.

The above experiments suggested that co-expression of the antibodybinding moiety/cell surface anchor fusion protein and the antibodytogether does not allow the anchor to be displayed at the cell surface.In the above experiments, both the antibody binding moiety/cell surfaceanchor fusion protein and antibody were expressed from nucleic acidmolecules operably linked to the strong AOX inducible promoter. It washypothesized that inducing expression of the antibody bindingmoiety/cell surface anchor fusion protein first, then after sufficientantibody binding moiety/cell surface anchor fusion protein had been madeand anchored to the cell surface, inhibiting expression of the antibodybinding moiety/cell surface anchor fusion protein and inducingexpression of the antibody, would enable the antibody that is made to becaptured at the cell surface by the antibody binding moiety/cell surfaceanchor fusion protein. Therefore, different promoters that would allowtemporal expression of the nucleic acid molecules encoding the antibodybinding moiety/cell surface anchor fusion protein and antibody weretested.

The GUT1 promoter is a promoter that is induced in cells grown in thepresence of glycerol and repressed when the cells are switched to amedium that lacks glycerol but contains dextrose. PCR was used toamplify the GUT1 promoter from genomic DNA of Pichia pastoris asBg/II/EcoRI fragment using primer SgutBglII ATTGAGATCT ACCCAATTTAGCAGCCTGCA TTCTC (SEQ ID NO:57) and primer 3gutEcoRI GTCAGAATTCATCTGTGGTA TAGTGTGAAA AAGTAG (SEQ ID NO:58). The PCR fragment was thencloned into the pCR2.1 TOPO vector, and then sequenced to confirm thesequence. The GUT1 promoter fragment was extracted from the pCR2.1 TOPOvector by Bg/II/EcoRI digest and cloned into pGLY4136 digested withBg/II/EcoRI to exchange the AOX1 promoter by the GUT1 promoter. Thenucleotide sequence of the GUT1 promoter including the Bg/I and EcoRIends is shown in SEQ ID NO:59.

The AOX promoter from the Protein A/SED1 fusion protein plasmid pGLY4136was replaced either by the PpGAPDH promoter resulting in plasmidpGLY4139 or the GUT1 promoter producing the plasmid pGLY4144 (FIG. 15).The PpGAPDH promoter is induced in dextrose and at about 80% of thatlevel in glycerol, while the GUT1 promoter is induced in glycerol andrepressed in dextrose. pGLY4139 was transfected into yGLY4134,expressing anti-Her2 antibody under control of the AOX promoter.Additionally, pGLY4144 has been transfected into strain yGLY5434(yGLY2696 transfected with pGLY4142), in which anti-Her2 expression isregulated by the GAPDH promoter.

Strain yGLY4134 transfected with pGLY4136, in which expression of theProtein A/SED1 fusion protein and the anti-Her2 antibody are bothregulated by the AOX promoter, was grown in 600 μL BMGY (glycerol ascarbon source) in a 96 deep well plate or 50 mL BMGY in a 250 ml, shakeflask for two days. The cells were collected by centrifugation and thesupernatant was discarded. The cells were induced by incubationovernight in 300 μL or 25 mL BMMY (methanol as carbon source) with PMTiinhibitor.

Strain yGLY4134 transfected with pGLY4139, in which expression of theProtein A/SED1 fusion protein is regulated by the PpGAPDH promoter andexpression of the anti-Her2 antibody regulated by the AOX promoter, wasgrown in BMGY (glycerol as carbon source) and induced in BMMY with PMTiinhibitor (methanol as carbon source).

Strain yGLY5434 transfected with pGLY4144, in which expression of theProtein A/SED1 fusion protein is regulated by the GUT1 promoter andexpression of the anti-Her2 antibody is regulated by the GAPDH promoter,was grown in BMGY (glycerol as carbon source) and induced in BMDY withPMTi inhibitor (dextrose as carbon source). Dextrose inhibitstranscription from the GUT1 promoter. After induction, all three strainswere labeled with goat anti human IgG (H+L)-Alexa 488 as described inExample 1. In general, growth can be between 1.5 days to 3 days andinduction between 1 to 2 days. Strains are usually grown for 2 days andthen induced for another 2 days: afterwards the analysis is done.

FIG. 16 illustrates the results of cell surface staining of the abovestrains. As was shown in Example 5, co-expression of the Protein A/SED1fusion protein and anti-Her2 antibody, both under the strong AOXpromoter (yGLY4134 transfected with pGLY4136) does not show any cellsurface labeling. Expression of the Protein A/SED1 fusion protein underthe GAPDH promoter during growth in glycerol and the expression ofanti-Her2 antibody regulated by the AOX promoter during induction withmethanol (yGLY4134 transfected with pGLY4139) shows some weak butvisible cell surface labeling. In this case the Protein A/SED1 fusionprotein is still expressed at some level during induction of theantibody because the GAPDH promoter is not completely repressed undermethanol induction conditions. However, expression of the Protein A/SED1fusion protein under the GUT1 promoter during growth in glycerolfollowed by induction of the anti-Her2 antibody regulated by the GAPDHpromoter during induction in dextrose (YGLY5434 transfected withpGLY4144) showed strong cell surface labeling. In this case, the ProteinA/SED 1 fusion protein was not expressed under antibody inductionconditions because the GUT1 promoter is completely repressed indextrose.

FIG. 17 is a chart that illustrates the expected expression patterns ofProtein A/SED1 fusion protein and antibody under the control ofdifferent combinations of promoters. Expression of the Protein A/SED1fusion protein and the antibody under the strong AOX promoter, which isrepressed in the glycerol growth phase and induced in the methanolinduction phase, led to no detectable cell surface display. Likely,co-expression leads to a Protein A/SED1 fusion protein—antibody complexin the ER, which does not secrete to the cell surface or is degraded.

Expression of the Protein A/SED1 fusion protein under the GAPDH promoterduring growth in glycerol and expression of the antibody under the AOXpromoter during induction in methanol resulted in weak cell surfacedisplay. In this case, the Protein A/SED1 fusion protein is stillexpressed at some level during induction of the antibody because theGAPDH promoter is not repressed completely under methanol inductionconditions. This means that under induction conditions, there might becomplex formation between the Protein A/SED1 fusion protein and theantibody in the ER, which then clogs the secretory pathway leading toonly a small amount of Protein A/SED1 fusion protein at the cellsurface.

Expression of the Protein A/SED1 fusion protein under the GUT1 promoterduring growth in glycerol followed by expression of the antibody underthe GAPDH promoter while simultaneously repressing expression of theProtein A/SED1 fusion protein during induction of antibody expressionwith dextrose led to strong cell surface display. Thus, when the ProteinA/SED1 fusion protein is expressed first and then completely repressedduring antibody induction, the Protein A/SED1 fusion protein is secretedto the cell wall where it can capture the antibody when it is secreted.Although the antibody is expressed at some level during Protein A/SED1fusion protein growth because the GAPDH promoter is not repressed underglycerol, the level of expression of the antibody appears to be lowenough to not interfere with the Protein A/SED1 fusion proteinsecretion.

To demonstrate that the cell surface display of whole antibody byProtein A/SED1 fusion protein regulated under the GUT1 promoter isfunctional for different antibodies, the anti-CD20 antibody expressingstrain yGLY5757 was also transfected with plasmid pGLY4144, whichencodes Protein A/SED1 fusion protein whose expression is regulated theGUT1 promoter. Strain yGLY5757 is strain yGLY2696 transfected with theplasmid pGLY4078. Plasmid pGLY4078 encodes the heavy and light chain ofthe anti-CD20 antibody under the regulation of the GAPDH promoter.

Strain yGLY5757 expressing the anti-CD20 antibody operably linked to theGAPDH promoter and transfected with pGLY4144 (encodes Protein A/SED1fusion protein under control of the GUT1 promoter) and strain yGLY5434expressing the anti-Her2 antibody operably linked to the GAPDH promotertransfected with pGLY4144 were grown in glycerol for Protein A/SED1fusion protein expression followed by induction in dextrose for antibodyexpression and secretion as described for FIG. 6. Strong cell surfacestaining was observed for both antibodies (FIG. 18). This demonstratesthat temporal regulation enables different antibodies and not just theanti-Her2 antibodies to be displayed on the yeast surface by an anchoredantibody binding moiety.

FIG. 19 shows the results of FACS sorting of the samples from FIG. 8.The anti-Her2 expressing strain yGLY5757 transfected with pGLY4144, theanti-CD20 expressing strain yGLY5434 transfected with pGLY4144 and theempty strain yGLY2696 transfected with pGLY4144 were grown in glyceroland then induced in dextrose. Cells were labeled with goat anti humanIgG (H+L)-Alexa 488 and analyzed by FACS sorting. As shown in FIG. 9,the empty strain without antibody expression displayed backgroundfluorescent staining while for three clones of the anti-CD20 expressingstrain, the fluorescence was shifted to the right showing cell surfacelabeling. The same was also seen for the anti-Her2 expressing strain.One clone of this strain showed no cell surface labeling, which could bea false positive from a transfection that does not express the antibodyor the anchor. These results demonstrate that the cells displaying wholeantibodies can be sorted using FACS sorting.

TABLE 1 BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: DescriptionSequence 1 PCR primer AGCGCTGACGCCCCCGAGGAGGAGGACCAC hPDI/UP1 2PCR primer CCTTAATTAATTACAGTTCATCATGCACAGCTTTCTGAT hPDI/LP-PacI CAT 3PCR primer ATGAATTCAGGC CATATCGGCCATTGTTTACTGTGCG PB248 CCCACAGTAG 4PCR primer ATGTTTA AACGTGAGGATTACTGGTGATGAAAGAC PB249 5 PCR primerAGACTAGTCTATTTGGAG ACATTGACGGATCCAC PB250 6 PCR primerATCTCGAGAGGCCATGCAGGCCAACCACAAGATGAAT PB251 CAAATTTTG 7 PCR primerGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTC PpPDI/UPi-1 8 PCR primerGACCTTGATAGTCACTTGGGACCTCAACCTCACC PpPDI/LPi-1 9 PCR primerCGCCAATGATGAGGATGCCTCTTCAAAGGTTGTG PpPDI/UPi-2 10 PCR primerCACAACCTTTGAAGAGGCATCCTCATCATTGGCG PpPDI/LPi-2 11 PCR primerGGCGATTGCATTCGCGAC TGTATC PpPDI-5′/UP 12 PCR primerCCTAGAGAGCGGTGG CCAAGATG hPDI-3′/LP 13 PCR primerGTGGCCACACCAGGGGGC ATGGAAC hPDI/UP 14 PCR primerCCTAGAGAGCGGTGG CCAAGATG hPDI-3′/LP 15 PCR primerAGCGCTGACGATGAAGTTGATGTGGATGGTACA GTAG hGRP94/UP1 16 PCR primerGGCCGGCCTTACAATTCATCATG TTCAGCTGTAGATTC hGRP94/LP1 17 PCR primerTGAACCCATCTGTAAATAGAATGC PMT1-KO1 18 PCR primerGTGTCACCTAAATCGTATGTGCCCATTTACTGGA PMT1-KO2 AGCTGCTAACC 19 PCR primerCTCCCTATAGTGAGTCGTATTCATCATTGTACTTT PMT1-KO3 GGTATATTGG 20 PCR primerTATTTGTACCTGCGTCCTGTTTGC PMT1-KO4 21 PCR primer CACATACGATTTAGGTGACACPR29 22 PCR primer AATACGACTCACTATAGGGAG PR32 23 PCR primerTGCTCTCCGCGTGCAATAGAAACT PMT4-KO1 24 PCR primerCTCCCTATAGTGAGTCGTATTCACAGTGTACCATCT PMT4-KO2 TTCATCTCC 25 PCR primerGTGTCACCTAAATCGTATGTGAACCTAACTCTAA PMT4-KO3 TTCTTCAAAGC 26 PCR primerACTAGGGTATATAATTCCCAAGGT PMT4-KO4 27 Pre-pro α-ATG AGA TTC CCA TCC ATC TTC ACT GCT mating factorGTT TTG TTC GCT GCT TCT TCT GCT TTG signal peptide GCT (ScαMTprepro)(DNA) 28 Pre-pro α- MRFPSIFTAVLFAASSALA mating factor signal peptide(protein) 29 Anti-Her2 GAGGTTCAGTTGGTTGAATCTGGAGGAGGATTGGTTCAHeavy chain ACCTGGTGGTTCTTTGAGATTGTCCTGTGCTGCTTCCG (VH + IgG1GTTTCAACATCAAGGACACTTACATCCACTGGGTTAGA constant region)CAAGCTCCAGGAAAGGGATTGGAGTGGGTTGCTAGAAT (DNA)CTACCCAACTAACGGTTACACAAGATACGCTGACTCCGTTAAGGGAAGATTCACTATCTCTGCTGACACTTCCAAGAACACTGCTTACTTGCAGATGAACTCCTTGAGAGCTGAGGATACTGCTGTTTACTACTGTTCCAGATGGGGTGGTGATGGTTTCTACGCTATGGACTACTGGGGTCAAGGAACTTTGGTTACTGTTTCCTCCGCTTCTACTAAGGGACCATCTGTTTTCCCATTGGCTCCATCTTCTAAGTCTACTTCCGGTGGTACTGCTGCTTTGGGATGTTTGGTTAAAGACTACTTCCCAGAGCCAGTTACTGTTTCTTGGAACTCCGGTGCTTTGACTTCTGGTGTTCACACTTTCCCAGCTGTTTTGCAATCTTCCGGTTTGTACTCTTTGTCCTCCGTTGTTACTGTTCCATCCTCTTCCTTGGGTACTCAGACTTACATCTGTAACGTTAACCACAAGCCATCCAACACTAAGGTTGACAAGAAGGTTGAGCCAAAGTCCTGTGACAAGACTCATACTTGTCCACCATGTCCAGCTCCAGAATTGTTGGGTGGTCCTTCCGTTTTTTTGTTCCCACCAAAGCCAAAGGACACTTTGATGATCTCCAGAACTCCAGAGGTTACATGTGTTGTTGTTGACGTTTCTCACGAGGACCCAGAGGTTAAGTTCAACTGGTACGTTGACGGTGTTGAAGTTCACAACGCTAAGACTAAGCCAAGAGAGGAGCAGTACAACTCCACTTACAGAGTTGTTTCCGTTTTGACTGTTTTGCACCAGGATTGGTTGAACGGAAAGGAGTACAAGTGTAAGGTTTCCAACAAGGCTTTGCCAGCTCCAATCGAAAAGACTATCTCCAAGGCTAAGGGTCAACCAAGAGAGCCACAGGTTTACACTTTGCCACCATCCAGAGATGAGTTGACTAAGAACCAGGTTTCCTTGACTTGTTTGGTTAAGGGATTCTACCCATCCGACATTGCTGTTGAATGGGAGTCTAACGGTCAACCAGAGAACAACTACAAGACTACTCCACCTGTTTTGGACTCTGACGGTTCCTTTTTCTTGTACTCCAAGTTGACTGTTGACAAGTCCAGATGGCAACAGGGTAACGTTTTCTCCTGTTCCGTTATGCATGAGGCTTTGCACAACCACTACACTCAAAAGTCCT TGTCTTTGTCCCCTGGTAAGTAA 30Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR Heavy chainQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK (VH + IgG1NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT constant region)LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY (protein)FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK31 Anti-Her2 light GACATCCAAATGACTCAATCCCCATCTTCTTTGTCTGC chain (VL +Kappa TTCCGTTGGTGACAGAGTTACTATCACTTGTAGAGCTT constant region)CCCAGGACGTTAATACTGCTGTTGCTTGGTATCAACAG (DNA)AAGCCAGGAAAGGCTCCAAAGTTGTTGATCTACTCCGCTTCCTTCTTGTACTCTGGTGTTCCATCCAGATTCTCTGGTTCCAGATCCGGTACTGACTTCACTTTGACTATCTCCTCCTTGCAACCAGAAGATTTCGCTACTTACTACTGTCAGCAGCACTACACTACTCCACCAACTTTCGGACAGGGTACTAAGGTTGAGATCAAGAGAACTGTTGCTGCTCCATCCGTTTTCATTTTCCCACCATCCGACGAACAGTTGAAGTCTGGTACAGCTTCCGTTGTTTGTTTGTTGAACAACTTCTACCCAAGAGAGGCTAAGGTTCAGTGGAAGGTTGACAACGCTTTGCAATCCGGTAACTCCCAAGAATCCGTTACTGAGCAAGACTCTAAGGACTCCACTTACTCCTTGTCCTCCACTTTGACTTTGTCCAAGGCTGATTACGAGAAGCACAAGGTTTACGCTTGTGAGGTTACACATCAGGGTTTGTCCTCCCCAGTTACTAAGTCCTTCAACAGAGGAGAGTGTTAA 32 Anti-Her2 lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ chain (VL + KappaKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTIS constant region)SLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 33Alpha amylase ATGGTTGCTT GGTGGTCCTT GTTCTTGTAC signal peptideGGATTGCAAG TTGCTGCTCC AGCTTTGGCT (from Aspergillus niger α-amylase)(DNA) 34 Alpha amylase MVAWWSLFLY GLQVAAPALA signal peptide(from Aspergillus niger α-amylase) 35 Anti-CD20GAGATCGTTT TGACACAGTC CCCAGCTACT Light chainTTGTCTTTGT CCCCAGGTGA AAGAGCTACA Variable RegionTTGTCCTGTA GAGCTTCCCA ATCTGTTTCC (DNA) TCCTACTTGG CTTGGTATCA ACAAAAGCCAGGACAGGCTC CAAGATTGTT GATCTACGAC GCTTCCAATA GAGCTACTGG TATCCCAGCTAGATTCTCTG GTTCTGGTTC CGGTACTGAC TTCACTTTGA CTATCTCTTC CTTGGAACCAGAGGACTTCG CTGTTTACTA CTGTCAGCAG AGATCCAATT GGCCATTGAC TTTCGGTGGTGGTACTAAGG TTGAGATCAA GCGTACGGTT GCTGCTCCTT CCGTTTTCAT TTTCCCACCATCCGACGAAC AATTGAAGTC TGGTACCCAA TTCGCCC 36 Anti-CD20EIVLTQSPAT LSLSPGERAT LSCRASQSVS Light chainSYLAWYQQKP GQAPRLLIYD ASNRATGIPA VariableRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ Region RSNWPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTQFA 37 Anti-CD20 GCTGTTCAGC TGGTTGAATC TGGTGGTGGAHeavy chain TTGGTTCAAC CTGGTAGATC CTTGAGATTG Variable RegionTCCTGTGCTG CTTCCGGTTT TACTTTCGGT (DNA) GACTACACTA TGCACTGGGT TAGACAAGCTCCAGGAAAGG GATTGGAATG GGTTTCCGGT ATTTCTTGGA ACTCCGGTTC CATTGGTTACGCTGATTCCG TTAAGGGAAG ATTCACTATC TCCAGAGACA ACGCTAAGAA CTCCTTGTACTTGCAGATGA ACTCCTTGAG AGCTGAGGAT ACTGCTTTGT ACTACTGTAC TAAGGACAACCAATACGGTT CTGGTTCCAC TTACGGATTG GGAGTTTGGG GACAGGGAAC TTTGGTTACTGTCTCGAGTG CTTCTACTAA GGGACCATCC GTTTTTCCAT TGGCTCCATC CTCTAAGTCTACTTCCGGTG GTACCCAATT CGCCC 38 Anti-CD20AVQLVESGGG LVQPGRSLRL SCAASGFTFG Heavy chainDYTMHWVRQA PGKGLEWVSG ISWNSGSIGY Variable RegionADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCTKDN QYGSGSTYGL GVWGQGTLVTVSSASTKGPS VFPLAPSSKS TSGGTQFA 39 human PDI GeneGACGCCCCCGAGGAGGAGGACCACGTCTTGGTGCTGCG (DNA)GAAAAGCAACTTCGCGGAGGCGCTGGCGGCCCACAAGTACCCGCCGGTGGAGTTCCATGCCCCCTGGTGTGGCCACTGCAAGGCTCTGGCCCCTGAGTATGCCAAAGCCGCTGGGAAGCTGAAGGCAGAAGGTTCCGAGATCAGGTTGGCCAAGGTGGACGCCACGGAGGAGTCTGACCTAGCCCAGCAGTACGGCGTGCGCGGCTATCCCACCATCAAGTTCTTCAGGAATGGAGACACGGCTTCCCCCAAGGAATATACAGCTGGCAGAGAGGCTGATGACATCGTGAACTGGCTGAAGAAGCGCACGGGCCCGGCTGCCACCACCCTGCCTGACGGCGCAGCTGCAGAGTCCTTGGTGGAGTCCAGCGAGGTGGCCGTCATCGGCTTCTTCAAGGACGTGGAGTCGGACTCTGCCAAGCAGTTTTTGCAGGCAGCAGAGGCCATCGATGACATACCATTTGGGATCACTTCCAACAGTGACGTGTTCTCCAAATACCAGCTCGACAAAGATGGGGTTGTCCTCTTTAAGAAGTTTGATGAAGGCCGGAACAACTTTGAAGGGGAGGTCACCAAGGAGAACCTGCTGGACTTTATCAAACACAACCAGCTGCCCCTTGTCATCGAGTTCACCGAGCAGACAGCCCCGAAGATTTTTGGAGGTGAAATCAAGACTCACATCCTGCTGTTCTTGCCCAAGAGTGTGTCTGACTATGACGGCAAACTGAGCAACTTCAAAACAGCAGCCGAGAGCTTCAAGGGCAAGATCCTGTTCATCTTCATCGACAGCGACCACACCGACAACCAGCGCATCCTCGAGTTCTTTGGCCTGAAGAAGGAAGAGTGCCCGGCCGTGCGCCTCATCACCTTGGAGGAGGAGATGACCAAGTACAAGCCCGAATCGGAGGAGCTGACGGCAGAGAGGATCACAGAGTTCTGCCACCGCTTCCTGGAGGGCAAAATCAAGCCCCACCTGATGAGCCAGGAGCTGCCGGAGGACTGGGACAAGCAGCCTGTCAAGGTGCTTGTTGGGAAGAACTTTGAAGACGTGGCTTTTGATGAGAAAAAAAACGTCTTTGTGGAGTTCTATGCCCCATGGTGTGGTCACTGCAAACAGTTGGCTCCCATTTGGGATAAACTGGGAGAGACGTACAAGGACCATGAGAACATCGTCATCGCCAAGATGGACTCGACTGCCAACGAGGTGGAGGCCGTCAAAGTGCACGGCTTCCCCACACTCGGGTTCTTTCCTGCCAGTGCCGACAGGACGGTCATTGATTACAACGGGGAACGCACGCTGGATGGTTTTAAGAAATTCCTAGAGAGCGGTGGCCAAGATGGGGCAGGGGATGTTGACGACCTCGAGGACCTCGAAGAAGCAGAGGAGCCAGACATGGAGGAAGACG ATGACCAGAAAGCTGTGAAAGATGAACTGTAA40 human PDI Gene DAPEEEDHVLVLRKSNFAEALAAHKYPPVEFHAPWCGH (protein)CKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHGFPTLGFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDVDDLEDLEEAEEPDMEEDDDQKAVHDEL 41 Pichia pastorisATGCAATTCAACTGGAATATTAAAACTGTGGCAAGTAT PDI1 GeneTTTGTCCGCTCTCACACTAGCACAAGCAAGTGATCAGG (DNA)AGGCTATTGCTCCAGAGGACTCTCATGTCGTCAAATTGACTGAAGCCACTTTTGAGTCTTTCATCACCAGTAATCCTCACGTTTTGGCAGAGTTTTTTGCCCCTTGGTGTGGTCACTGTAAGAAGTTGGGCCCTGAACTTGTTTCTGCTGCCGAGATCTTAAAGGACAATGAGCAGGTTAAGATTGCTCAAATTGATTGTACGGAGGAGAAGGAATTATGTCAAGGCTACGAAATTAAAGGGTATCCTACTTTGAAGGTGTTCCATGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTCAAAGACAGAGCCAAAGCATTGTCAGCTATATGCTAAAGCAGAGTTTACCCCCTGTCAGTGAAATCAATGCAACCAAAGATTTAGACGACACAATCGCCGAGGCAAAAGAGCCCGTGATTGTGCAAGTACTACCGGAAGATGCATCCAACTTGGAATCTAACACCACATTTTACGGAGTTGCCGGTACTCTCAGAGAGAAATTCACTTTTGTCTCCACTAAGTCTACTGATTATGCCAAAAAATACACTAGCGACTCGACTCCTGCCTATTTGCTTGTCAGACCTGGCGAGGAACCTAGTGTTTACTCTGGTGAGGAGTTAGATGAGACTCATTTGGTGCACTGGATTGATATTGAGTCCAAACCTCTATTTGGAGACATTGACGGATCCACCTTCAAATCATATGCTGAAGCTAACATCCCTTTAGCCTACTATTTCTATGAGAACGAAGAACAACGTGCTGCTGCTGCCGATATTATTAAACCTTTTGCTAAAGAGCAACGTGGCAAAATTAACTTTGTTGGCTTAGATGCCGTTAAATTCGGTAAGCATGCCAAGAACTTAAACATGGATGAAGAGAAACTCCCTCTATTTGTCATTCATGATTTGGTGAGCAACAAGAAGTTTGGAGTTCCTCAAGACCAAGAATTGACGAACAAAGATGTGACCGAGCTGATTGAGAAATTCATCGCAGGAGAGGCAGAACCAATTGTGAAATCAGAGCCAATTCCAGAAATTCAAGAAGAGAAAGTCTTCAAGCTAGTCGGAAAGGCCCACGATGAAGTTGTCTTCGATGAATCTAAAGATGTTCTAGTCAAGTACTACGCCCCTTGGTGTGGTCACTGTAAGAGAATGGCTCCTGCTTATGAGGAATTGGCTACTCTTTACGCCAATGATGAGGATGCCTCTTCAAAGGTTGTGATTGCAAAACTTGATCACACTTTGAACGATGTCGACAACGTTGATATTCAAGGTTATCCTACTTTGATCCTTTATCCAGCTGGTGATAAATCCAATCCTCAACTGTATGATGGATCTCGTGACCTAGAATCATTGGCTGAGTTTGTAAAGGAGAGAGGAACCCACAAAGTGGATGCCCTAGCACTCAGACCAGTCGAGGAAGAAAAGGAAGCTGAAGAAGAAGCTGAAAGTGAGGCAGACGCTCACGACGAGCTTTAA 42 Pichia pastorisMQFNWNIKTVASILSALTLAQASDQEAIAPEDSHVVKL PDI1 GeneTEATFESFITSNPHVLAEFFAPWCGHCKKLGPELVSAA (protein)EILKDNEQVKIAQIDCTEEKELCQGYEIKGYPTLKVFHGEVEVPSDYQGQRQSQSIVSYMLKQSLPPVSEINATKDLDDTIAEAKEPVIVQVLPEDASNLESNTTFYGVAGTLREKFTFVSTKSTDYAKKYTSDSTPAYLLVRPGEEPSVYSGEELDETHLVHWIDIESKPLFGDIDGSTFKSYAEANIPLAYYFYENEEQRAAAADIIKPFAKEQRGKINFVGLDAVKFGKHAKNLNMDEEKLPLFVIHDLVSNKKFGVPQDQELTNKDVTELIEKFIAGEAEPIVKSEPIPEIQEEKVFKLVGKAHDEVVFDESKDVLVKYYAPWCGHCKRMAPAYEELATLYANDEDASSKVVIAKLDHTLNDVDNVDIQGYPTLILYPAGDKSNPQLYDGSRDLESLAEFVKERGTHKVDALAL RPVEEEKEAEEEAESEADAHDEL 43human GRP94 Gene GATGATGAAGTTGACGTTGACGGTACTGTTGAAGAGGA (DNA)CTTGGGAAAGTCTAGAGAGGGTTCCAGAACTGACGACGAAGTTGTTCAGAGAGAGGAAGAGGCTATTCAGTTGGACGGATTGAACGCTTCCCAAATCAGAGAGTTGAGAGAGAAGTCCGAGAAGTTCGCTTTCCAAGCTGAGGTTAACAGAATGATGAAATTGATTATCAACTCCTTGTACAAGAACAAAGAGATTTTCTTGAGAGAGTTGATCTCTAACGCTTCTGACGCTTTGGACAAGATCAGATTGATCTCCTTGACTGACGAAAACGCTTTGTCCGGTAACGAAGAGTTGACTGTTAAGATCAAGTGTGACAAAGAGAAGAACTTGTTGCACGTTACTGACACTGGTGTTGGAATGACTAGAGAAGAGTTGGTTAAGAACTTGGGTACTATCGCTAAGTCTGGTACTTCCGAGTTCTTGAACAAGATGACTGAGGCTCAAGAAGATGGTCAATCCACTTCCGAGTTGATTGGTCAGTTCGGTGTTGGTTTCTACTCCGCTTTCTTGGTTGCTGACAAGGTTATCGTTACTTCCAAGCACAACAACGACACTCAACACATTTGGGAATCCGATTCCAACGAGTTCTCCGTTATTGCTGACCCAAGAGGTAACACTTTGGGTAGAGGTACTACTATCACTTTGGTTTTGAAAGAAGAGGCTTCCGACTACTTGGAGTTGGACACTATCAAGAACTTGGTTAAGAAGTACTCCCAGTTCATCAACTTCCCAATCTATGTTTGGTCCTCCAAGACTGAGACTGTTGAGGAACCAATGGAAGAAGAAGAGGCTGCTAAAGAAGAGAAAGAGGAATCTGACGACGAGGCTGCTGTTGAAGAAGAGGAAGAAGAAAAGAAGCCAAAGACTAAGAAGGTTGAAAAGACTGTTTGGGACTGGGAGCTTATGAACGACATCAAGCCAATTTGGCAGAGACCATCCAAAGAGGTTGAGGAGGACGAGTACAAGGCTTTCTACAAGTCCTTCTCCAAAGAATCCGATGACCCAATGGCTTACATCCACTTCACTGCTGAGGGTGAAGTTACTTTCAAGTCCATCTTGTTCGTTCCAACTTCTGCTCCAAGAGGATTGTTCGACGAGTACGGTTCTAAGAAGTCCGACTACATCAAACTTTATGTTAGAAGAGTTTTCATCACTGACGACTTCCACGATATGATGCCAAAGTACTTGAACTTCGTTAAGGGTGTTGTTGATTCCGATGACTTGCCATTGAACGTTTCCAGAGAGACTTTGCAGCAGCACAAGTTGTTGAAGGTTATCAGAAAGAAACTTGTTAGAAAGACTTTGGACATGATCAAGAAGATCGCTGACGACAAGTACAACGACACTTTCTGGAAAGAGTTCGGAACTAACATCAAGTTGGGTGTTATTGAGGACCACTCCAACAGAACTAGATTGGCTAAGTTGTTGAGATTCCAGTCCTCTCATCACCCAACTGACATCACTTCCTTGGACCAGTACGTTGAGAGAATGAAAGAGAAGCAGGACAAAATCTACTTCATGGCTGGTTCCTCTAGAAAAGAGGCTGAATCCTCCCCATTCGTTGAGAGATTGTTGAAGAAGGGTTACGAGGTTATCTACTTGACTGAGCCAGTTGACGAGTACTGTATCCAGGCTTTGCCAGAGTTTGACGGAAAGAGATTCCAGAACGTTGCTAAAGAGGGTGTTAAGTTCGACGAATCCGAAAAGACTAAAGAATCCAGAGAGGCTGTTGAGAAAGAGTTCGAGCCATTGTTGAACTGGATGAAGGACAAGGCTTTGAAGGACAAGATCGAGAAGGCTGTTGTTTCCCAGAGATTGACTGAATCCCCATGTGCTTTGGTTGCTTCCCAATACGGATGGAGTGGTAACATGGAAAGAATCATGAAGGCTCAGGCTTACCAAACTGGAAAGGACATCTCCACTAACTACTACGCTTCCCAGAAGAAAACTTTCGAGATCAACCCAAGACACCCATTGATCAGAGACATGTTGAGAAGAATCAAAGAGGACGAGGACGACAAGACTGTTTTGGATTTGGCTGTTGTTTTGTTCGAGACTGCTACTTTGAGATCCGGTTACTTGTTGCCAGACACTAAGGCTTACGGTGACAGAATCGAGAGAATGTTGAGATTGTCCTTGAACATTGACCCAGACGCTAAGGTTGAAGAAGAACCAGAAGAAGAGCCAGAGGAAACTGCTGAAGATACTACTGAGGACACTGAACAAGACGAGGACGAAGAGATGGATGTTGGTACTGACGAAGAGGAAGAGACAGCAAA GGAATCCACTGCTGAACACGACGAGTTGTAA44 human GRP94 Gene DDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLD (protein)GLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEE MDVGTDEEEETAKESTAEHDEL 45Protein A GAATTCGAAACGATGAGATTCCCATCCATCTTCACTGC fusion proteinTGTTTTGTTCGCTGCTTCTTCTGCTTTGGCGGCCGCTA (apre-5xBD-Htag) asATGCTGCTCAACACGACGAAGCTCAACAGAACGCTTTC EcoRI/SalITACCAGGTTTTGAACATGCCAAACTTGAACGCTGACCA fragment,GAGGAATGGTTTCATCCAGTCCTTGAAGGATGACCCAT including alphaCTCAATCCGCTAACGTTTTGGGTGAAGCTCAGAAGTTG MF pre signalAACGACAGTCAAGCTCCTAAGGCTGATGCTCAACAAAA sequenceCAACTTCAACAAGGACCAGCAATCTGCTTTCTACGAAA (underlined), 5TCTTGAATATGCCTAATTTGAACGAGGCTCAGAGAAAT Fc bindingGGATTCATTCAATCTTTGAAAGACGACCCATCCCAGTC domains, and aTACTAATGTTTTGGGAGAGGCTAAGAAACTTAATGAAA HA and 9 xGTCAGGCTCCTAAAGCTGACAACAACTTTAACAAAGAG HIS tag at theCAGCAGAACGCTTTTTATGAGATTCTTAACATGCCTAA C-terminus.CTTGAACGAAGAGCAAAGAAACGGTTTTATTCAATCATTGAAGGACGATCCTTCACAGTCTGCTAACTTGTTGTCCGAGGCTAAAAAGTTGAACGAATCTCAGGCTCCTAAGGCTGATAATAAGTTCAACAAAGAACAACAAAATGCTTTCTACGAGATTTTGCACTTGCCAAATTTGAATGAGGAACAGAGAAACGGTTTTATTCAGTCATTGAAGGATGACCCTTCCCAATCTGCTAATTTGTTGGCTGAAGCTAAGAAATTGAACGACGCTCAGGCTCCAAAAGCTGATAACAAATTCAACAAAGAGCAACAGAACGCTTTCTACGAAATCTTGCATTTGCCAAACTTGACAGAAGAGCAGAGAAACGGATTCATTCAGTCTTTGAAGGATGACCCTTCCGTTTCCAAAGAGATTTTGGCTGAGGCTAAAAAGTTGAATGATGCTCAAGCTCCAAAAGGTGGTGGTTACCCATACGATGTTCCAGATTACGCTGGAGGTCATCATCATCACCACCATCACCATCATGGT GGTGTCGAC 46 Protein AMRFPSIFTAVLFAASSALAAANAAQHDEAQQNAFYQVL fusion protein;NMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQ alpha-MF-pre-APKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQ signal isSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNA underlinedFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKGGGYPYDVPDYAGGHHHHHHHHHGGVD 47 alpha-amylase-CGGAATTCacgatggtcgcttggtggtctttgtttctg ProtAZZ/up:tacggtcttcaggtcgctgcacctgctttggctTCTGGTGGTGTTACTCCAGCTGCTAACGCTGCTCAACACG 48 HA-ProtAZZ-GCCTCGAGAGCGTAGTCTGGAACATCGTATGGGTAACC Xho1ZZ/1p: ACCACCAGCATC 49DNA sequence GAATTCacgatggtcgcttggtggtctttgtttctgta of the ZZ-cggtcttcaggtcgctgcacctgctttggctTCTGGTG domain asGTGTTACTCCAGCTGCTAACGCTGCTCAACACGATGAA EcoRI/XhoIGCTGTTGACAACAAGTTCAACAAAGAGCAGCAGAACGC fragment:TTTCTACGAGATCTTGCACTTGCCAAACTTGAACGAAG Alpha-amylaseAGCAAAGAAACGCTTTCATCCAGTCCTTGAAGGATGAC sequenceCCATCTCAATCCGCTAACTTGTTGGCTGAGGCTAAGAA underlinedGTTGAACGACGCTCAAGCTCCAAAGGTCGACAATAAGTTTAACAAAGAACAACAAAATGCCTTCTACGAAATTCTGCATCTGCCCAACCTTAACGAGGAACAGAGAAACGCCTTCATTCAGAGTTTGAAGGACGATCCTTCCCAGTCTGCTAATTTGCTTGCCGAAGCCAAGAAATTGAATGATGCCCAGGCTCCAAAAGTTGATGCTGGTGGTGGTTACCCATACGA TGTTCCAGACTACGCTCTCGAG 50 ProteinMVAWWSLFLYGLQVAAPALASGGVTPAANAAQHDEAVD sequence of theNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQ ZZ-domain:SANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLP Alpha-amylaseNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK leader is VDAGGGYPYDVPDYALEunderlined 51 5Ecoapp: AACGGAATTCATGAGATTTCCTTCAATTTTTAC 52 3HtagSalCGATGTCGACGTGATGGTGATGGTGGTGATGATGATGA CCACC 53 DNA sequenceGAATTCATGAGATTTCCTTCAATTTTTACTGCTGTTTT of theATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACA FcRIII(LF) asCTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAA EcoRI/SalIGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGA fragment:TGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGCTGGAATGAGAACTGAGGACTTGCCAAAGGCTGTTGTTTTCTTGGAGCCACAGTGGTACAGAGTTTTGGAGAAGGATTCCGTTACTTTGAAGTGTCAGGGAGCTTACTCTCCAGAAGATAACTCCACTCAGTGGTTCCACAACGAATCCTTGATTTCTTCTCAGGCTTCCTCCTACTTCATTGACGCTGCTACTGTTGACGATTCCGGTGAGTACAGATGTCAGACTAACTTGTCCACTTTGTCCGACCCAGTTCAATTGGAGGTTCACATCGGTTGGTTGTTGTTGCAAGCTCCAAGATGGGTTTTCAAGGAGGAGGACCCAATTCATTTGAGATGTCACTCTTGGAAGAACACTGCTTTGCACAAAGTTACTTACTTGCAGAACGGAAAGGGTAGAAAGTATTTCCACCACAACTCCGACTTCTACATCCCAAAGGCTACTTTGAAGGATTCCGGTTCCTACTTCTGTAGAGGATTGTTCGGTTCCAAGAACGTTTCTTCCGAGACTGTTAACATCACTATCACTCAGGGATTGGCTGTTTCCACTATCTCTTCCTTCTTCCCACCAGGTTATCAAGGTGGTGGTCATCATCATCACCACCATCACCATC ACGTCGAC 54 ProteinMRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAV sequence of theIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAK FcRIII(LF) withEEGVSLEKRAGMRTEDLPKAVVFLEPQWYRVLEKDSVT alpha MF preLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVD signal sequenceDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKE and HIS Tag:EDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAV STISSFFPPGYQGGGHHHHHHHHHVD 55DNA sequence GAATTCATGAGATTTCCTTCAATTTTTACTGCTGTTTT of the FcRIasATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACA EcoRI/SalICTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAA fragment:GCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGCTGATACTACTAAGGCTGTTATCACTTTGCAACCACCATGGGTTTCCGTTTTCCAGGAGGAGACTGTTACTTTGCACTGTGAGGTTTTGCATTTGCCTGGTTCCTCTTCCACTCAGTGGTTCTTGAACGGTACTGCTACTCAAACTTCCACTCCATCCTACAGAATTACTTCCGCTTCCGTTAACGATTCTGGTGAGTACAGATGTCAGAGAGGATTGTCTGGTAGATCCGACCCAATTCAGTTGGAGATTCACAGAGGATGGTTGTTGTTGCAGGTTTCCTCCAGAGTTTTCACTGAGGGTGAACCATTGGCTTTGAGATGTCACGCTTGGAAGGACAAGTTGGTTTACAACGTTTTGTACTACAGAAACGGAAAGGCTTTCAAGTTCTTCCACTGGAACTCCAACTTGACTATCTTGAAAACTAACATCTCCCACAACGGTACTTACCACTGTTCTGGAATGGGAAAGCACAGATACACTTCCGCTGGTATCTCCGTTACTGTTAAGGAGTTGTTCCCAGCTCCAGTTTTGAACGCTTCCGTTACTTCTCCATTGTTGGAGGGAAACTTGGTTACTTTGTCCTGTGAGACTAAATTGTTGTTGCAAAGACCAGGATTGCAGTTGTACTTCTCCTTCTACATGGGTTCCAAGACTTTGAGAGGTAGAAACACTTCCTCCGAGTACCAAATCTTGACTGCTAGAAGAGAGGATTCCGGTTTGTACTGGTGTGAAGCTGCTACTGAGGACGGTAACGTTTTGAAGAGATCCCCAGAGTTGGAGTTGCAAGTTTTGGGATTGCAATTGCCAACTCCAGGTGGTGGTCATCATCATCACCACC ATCACCATCACGTCGAC 56 ProteinMRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAV sequence of theIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAK FcRI with alphaEEGVSLEKRADTTKAVITLQPPWVSVFQEETVTLHCEV MF pre signalLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEY sequence andRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLA HIS Tag:LRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRS PELELQVLGLQLPTPGGGHHHHHHHHHVD 575gutBglII: ATTGAGATCTACCCAATTTAGCAGCCTGCATTCTC 58 3gutEcoRI:GTCAGAATTCATCTGTGGTATAGTGTGAAAAAGTAG 59 DNA sequenceAGATCTACCCAATTTAGCAGCCTGCATTCTCTTGATTT GUT1 promoterTATGGGGGAAACTAACAATAGTGTTGCCTTGATTTTAAGTGGCATTGTTCTTTGAAATCGAAATTGGGGATAACGTCATACCGAAAGGTAAACAACTTCGGGGAATTGCCCTGGTTAAACATTTATTAAGCGAGATAAATAGGGGATAGCGAGATAGGGGGCGGAGAAGAAGAAGGGTGTTAAATTGCTGAAATCTCTCAATCTGGAAGAAACGGAATAAATTAACTCCTTCCTGAGATAATAAGATCCGACTCTGCTATGACCCCACACGGTACTGACCTCGGCATACCCCATTGGATCTGGTGCGAAGCAACAGGTCCTGAAACCTTTATCACGTGTAGTAGATTGACCTTCCAGCAAAAAAAGGCATTATATATTTTGTTGTTGAAGGGGTGAGGGGAGGTGCAGGTGGTTCTTTTATTCGTCTTGTAGTTAATTTTCCCGGGGTTGCGGAGCGTCAAAAGTTTGCCCGATCTGATAGCTTGCAAGATGCCACCGCTTATCCAACGCACTTCAGAGAGCTTGCCGTAGAAAGAACGTTTTCCTCGTAGTATTCCAGCACTTCATGGTGAAGTCGCTATTTCACCGAAGGGGGGGTATTAAGGTTGCGCACCCCCTCCCCACACCCCAGAATCGTTTATTGGCTGGGTTCAATGGCGTTTGAGTTAGCACATTTTTTCCTTAAACACCCTCCAAACACGGATAAAAATGCATGTGCATCCTGAAACTGGTAGAGATGCGTACTCCGTGCTCCGATAATAACAGTGGTGTTGGGGTTGCTGTTAGCTCACGCACTCCGTTTTTTTTTCAACCAGCAAAATTCGATGGGGAGAAACTTGGGGTACTTTGCCGACTCCTCCACCATACTGGTATATAAATAATACTCGCCCACTTTTCGTTTGCTGCTTTTATATTTCAAGGACTGAAAAAGACTCTTCTTCTACTTTTTC ACACTATACCACAGATGAATTC 60S. cerevisiae VDQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPES SEDI (withoutDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTN endogenousGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPT leader sequenceTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTTLTITDCPCTIEKPTTTSTTEYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTTLTITDCPCTIEKSEAPESSVPVTESKGTTTKETGVTTKQTTANPSLTVSTVVPVSSSASSHSVVINSNGA NVVVPGALGLAGVAMLFL 61S. cerevisiae GTCGACCAATTCTCTAACTCTACTTCCGCTTCCTCTAC SEDI DNATGACGTTACTTCCTCCTCCTCTATTTCTACTTCCTCCG sequenceGTTCCGTTACTATTACTTCCTCTGAGGCTCCAGAATCTGACAACGGTACTTCTACTGCTGCTCCAACTGAAACTTCTACTGAGGCTCCTACTACTGCTATTCCAACTAACGGAACTTCCACAGAGGCTCCAACAACAGCTATCCCTACAAACGGTACATCCACTGAAGCTCCTACTGACACTACTACAGAAGCTCCAACTACTGCTTTGCCTACTAATGGTACATCAACAGAGGCTCCTACAGATACAACAACTGAAGCTCCAACAACTGGATTGCCAACAAACGGTACTACTTCTGCTTTCCCACCAACTACTTCCTTGCCACCATCCAACACTACTACTACTCCACCATACAACCCATCCACTGACTACACTACTGACTACACAGTTGTTACTGAGTACACTACTTACTGTCCAGAGCCAACTACTTTCACAACAAACGGAAAGACTTACACTGTTACTGAGCCTACTACTTTGACTATCACTGACTGTCCATGTACTATCGAGAAGCCAACTACTACTTCCACTACAGAGTATACTGTTGTTACAGAATACACAACATATTGTCCTGAGCCAACAACATTCACTACTAATGGAAAAACATACACAGTTACAGAACCAACTACATTGACAATTACAGATTGTCCTTGTACAATTGAGAAGTCCGAGGCTCCTGAATCTTCTGTTCCAGTTACTGAATCCAAGGGTACTACTACTAAAGAAACTGGTGTTACTACTAAGCAGACTACTGCTAACCCATCCTTGACTGTTTCCACTGTTGTTCCAGTTTCTTCCTCTGCTTCTTCCCACTCCGTTGTTATCAACTCCAACGGTGCTAACGTTGTTGTTCCTGGTGCTTTGGGATTGGCTGGTGT TGCTATGTTGTTCTTGTAA

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1-10. (canceled)
 11. A method of producing eukaryote host cells thatproduce an immunoglobulin having a VH domain and a VL domain and havingan antigen binding site with binding specificity for an antigen ofinterest, the method comprising: (a) providing a library of eukaryotehost cells displaying on their surface an immunoglobulin comprising a VHdomain and a VL domain, wherein the library is created by: (i) providingeukaryote host cells that express a capture moiety comprising a cellsurface anchoring protein fused to a moiety capable of binding to animmunoglobulin wherein expression of the capture moiety is effected by afirst regulatable promoter; and (ii) transfecting the host cells with alibrary of nucleic acid molecules encoding a genetically diversepopulation of immunoglobulins, wherein the VH domains of the geneticallydiverse population of immunoglobulins are biased for one or more VH genefamilies and wherein expression of at least one of the heavy or lightchains of the immunoglobulins is effected by a second regulatablepromoter to produce a plurality of host cells, each expressing animmunoglobulin; (b) inducing expression of the capture moiety in thehost cells for a time sufficient to produce the capture moiety on thesurface of the host cells; and (c) inhibiting expression of the capturemoiety and inducing expression of the library of nucleic acid moleculesequences in the host cells, whereby each host cell displays animmunoglobulin at the surface thereof to produce the host cells thatproduce the immunoglobulin having a VH domain and a VL domain and havingthe antigen binding site with binding specificity for the antigen ofinterest.
 12. The method of claim 11, wherein the immunoglobulincomprises a synthetic human immunoglobulin VH domain and a synthetichuman immunoglobulin VL domain and wherein the synthetic humanimmunoglobulin VH domain and the synthetic human immunoglobulin VLdomain comprise framework regions and hypervariable loops, wherein theframework regions and first two hypervariable loops of both the VHdomain and VL domain are essentially human germ line, and wherein the VHdomain and VL domain have altered CDR3 loops.
 13. The method of claim12, wherein in addition to having altered CDR3 loops the human syntheticimmunoglobulin VH and VL domains contain mutations in other CDR loops.14. The method of claim 12, wherein each human synthetic immunoglobulinVH domain CDR loop is of random sequence.
 15. The method of claim 12,wherein human synthetic immunoglobulin VH domain CDR loops are of knowncanonical structures and incorporate random sequence elements.
 16. Themethod of claim 11 wherein the method further includes (d) identifyinghost cells in the plurality of host cells that display immunoglobulinsthereon that has a binding specificity for the antigen of interest bycontacting the plurality of host cells with the antigen of interest anddetecting the host cells that have the antigen of interest bound to theimmunoglobulin displayed thereon to produce the host cells that producethe immunoglobulin having a VH domain and a VL domain and having theantigen binding site with binding specificity for the antigen ofinterest
 17. The method of claim 11, wherein the antibody is selectedfrom the group consisting of IgG, IgA, IgM, and IgE.
 18. The method ofclaim 11, wherein the binding moiety binds to the Fc region of theimmunoglobulin.
 19. The method of claim 11, wherein the binding moietyis selected from the group consisting of protein A, protein A ZZ domain,protein G, and protein L.
 20. The method of claim 11, wherein the cellsurface anchoring protein is a GPI protein. 21-24. (canceled)